Dac hyp compositions

ABSTRACT

The present disclosure relates to compositions of daclizumab suitable for subcutaneous administration and methods of manufacturing thereof.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional under 35 U.S.C. §121 of U.S.application Ser. No. 14/601,909, filed Jan. 21, 2015, which is adivisional under 35 U.S.C. §121 of U.S. application Ser. No. 13/481,081,filed May 25, 2012, which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application No. 61/490,998, filed May 27, 2011, thecontents of all of which are incorporated herein in their entireties byreference thereto.

2. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 25, 2012, isnamed 386003US.txt and is 34,217 bytes in size.

3. BACKGROUND

Daclizumab (DAC) is a humanized IgG₁ monoclonal antibody that binds tothe alpha subunit (CD25 or Tac) of the human high-affinity interleukin-2(IL-2) receptor, which is expressed on the surface of activated, but notresting, T- and B-lymphocytes. When bound to CD25 on the activatedcells, DAC blocks the formation of the high affinity IL-2 receptorcomplex, thereby blocking IL-2-induced proliferation of the activatedcells.

As measured in direct binding assays on PHA blasts, DAC binds to CD25with an approximate binding affinity (K_(D)) of 0.3 nM, and inhibits theproliferation of PHA blasts in a dose-dependent manner (Hakimi et al.,1993, J. Immunol. 151(2):1075-85). At a suboptimal dose of IL-2 (2.5ng/mL), 15 nM DAC inhibits proliferation of IL-2-dependent cell lineKit225/K6 by 50% (Pilson et al., 1997, J. Immunol. 159(3):1543-56). Inan IL2-dependent antigen-induced T-cell proliferation assay, 50%inhibition of proliferation was observed with DAC in the range of 0.5-1μg/mL (3-7 nM) (Junghans et al., 1990, Cancer Res. 50(5):1495-502).

A version of DAC was previously marketed for the treatment of acuteallograft rejection in renal transplant patients as an adjunct to animmunosuppressive regimen that includes cyclosporine and corticosteroidsby Hoffman-La Roche, Inc. under the tradename ZENAPAX™. ZENAPAX wassupplied as a concentrate for further dilution and intravenousadministration. Each vial of concentrate contained 5 mL of a solutioncontaining 5 mg/mL DAC, 3.6 mg/mL sodium phosphate monobasicmonohydrate, 11 mg/mL sodium phosphate dibasic heptahydrate, 4.6 mg/mLsodium chloride, 0.2 mg/mL polysorbate 80 and HCl and/or NaOH sufficientto adjust the pH to pH 6.9. The recommended dose for both adult andpediatric patients was 1.0 mg/kg, prepared by diluting the calculatedvolume of 25 mg/5 mL ZENAPAX concentrate with 50 mL sterile 0.9% sodiumchloride solution and administering intravenously via a peripheral orcentral vein over a 15-minute period.

DAC has also shown efficacy in the treatment of uveitis (Nussenblatt etal., 2004, FOCIS 2004 meeting; July 18-23, Montreal, QC. Abstract 4688;Nussenblatt et al., 2003, J. Autoimmun. 21:283-93) and multiplesclerosis (see, e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci.USA 101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S.Pat. No. 7,258,859), and is currently the subject of ongoing clinicaltrials for the treatment of multiple sclerosis. Although DAC has provento be safe and effective, high concentration, liquid formulations thathave long shelf lives and that can be conveniently administered withoutfurther formulation or manipulation, as well as new daclizumab moleculesthat have improved properties, such as enhanced safety, as compared toZENAPAX DAC, would be desirable.

4. SUMMARY

As mentioned in the Background Section, daclizumab is a humanized IgG₁antibody that specifically binds the alpha subunit (also referred to asCD25 or Tac) of the human interleukin-2 receptor (IL-2R), which is animportant mediator of lymphocyte activation. A version of daclizumabpreviously marketed by Hoffman-La Roche under the tradename ZENAPAX™ hasdemonstrated safety and efficacy in the treatment of renal allograftrejection when used as an adjunct to an immunosuppressive regimenincluding cyclosporine and corticosteroids (see, e.g., the EuropeanMedicines Agency (“EMEA”) market authorization for ZENAPAX), and hasalso demonstrated efficacy in the treatment of multiple sclerosis (see,e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci. USA101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S. Pat.No. 7,258,859). According to EMEA, ZENAPAX DAC is expressed in GS-NS0(murine myeloma) cells and purified using a process that involvesQ-Sepharose chromatography, S-Sepharose chromatography, diafiltration,Q-Sepharose II chromatography, ultrafiltration, S-300 gel filtrationchromatography and ultrafiltration. It has now been discovered thatdaclizumab expressed in an NS0 cell line that has been adapted to growin serum-free, cholesterol-free and other animal product-free media andthat is isolated by a different process has characteristics andproperties that are different from, and in some instances, superior to,ZENAPAX daclizumab (“ZENAPAX DAC”). This new daclizumab, referred toherein as DAC HYP, has: a different isoform profile than ZENAPAX DAC (asdetermined via cation exchange chromatography); a different N-linkedglycosylation profile than ZENAPAX DAC, even though both forms ofdaclizumab are expressed in NS0 cells; and less ADCC cytotoxicity thanZENAPAX DAC in biological assays.

For example, isoforms of daclizumab are possible due to heterogeneity atthe heavy chain N- and C-termini. The amino acid sequence of the matureV_(H) chain of daclizumab begins at position 20 of the amino acidsequence shown in FIG. 2 (SEQ ID NO:4). The N-terminal glutamine (Q) ofthe mature V_(H) chain (in bold, underlined text in FIG. 2) can cyclize,forming pyroglutamate (pE). In some instances, the signal peptidesequence may truncate, leaving a valine-histidine-serine (VHS) sequenceattached to the N-terminal glutamine residue of the mature V_(H) chain.Because each daclizumab molecule contains two V_(H) chains, the variousN-terminal isoforms of daclizumab can include forms containing: (1) twoglutamine residues (Q/Q); (2) one glutamine residue and one VHS sequence(Q/VHS or VHS/Q); (3) two VHS sequences (VHS/VHS); (4) one glutamineresidue and one pyroglutamate residue (Q/pE or pE/Q); (5) onepyroglutamate residue and one VHS sequence (pE/VHS or VHS/pE); and (6)two pyroglutamate residues (pE/pE). Different C-terminal isoforms alsoare possible, which contain either 0, 1 or 2 C-terminal lysine (K)residues (0K, 1K or 2K), resulting in a complex isoform profile.

Quite surprisingly, while the N-terminal glutamines of the V_(H) chainsof ZENAPAX DAC are completely cyclized to pyroglutamate, completecyclization is not achieved for DAC HYP. As a consequence, the cationexchange chromatogram of DAC HYP is characterized by a pE/Q isoform peakand a Q/VHS isoform peak. While not intending to be bound by any theory,it is believed that these unique pE/Q and Q/VHS isoforms may beinfluenced by the leader sequence used to express DAC HYP. Accordingly,in one aspect, the present disclosure provides daclizumab compositionsin which the pE/Q isoform ranges from 3%-17%, from 3%-15%, from 5%-15%,more preferably from 5%-12% or 7%-12% of the N-terminal isoforms, and/orin which the Q/VHS isoform ranges from 1%-15%, more preferably 3%-12% ofthe N-terminal isoforms, as determined by cation exchangechromatography.

In some embodiments, the daclizumab composition is characterized by acation exchange chromatography profile that is substantially similar toFIG. 18 or the DAC HYP profile of FIG. 23.

Daclizumab has N-linked oligosaccharides attached to heavy chain residueAsn 296. When these N-linked oligosaccharides are released using amidasePNGaseF and analyzed via HPLC, DAC HYP exhibits a glycosylation profiledifferent from ZENAPAX DAC, despite the fact that both are recombinantlyproduced in NS0 cell lines. Indeed, the glycosylation profile of DAC HYPis unusually homogeneous. Referring to the upper panel of FIG. 21, theglycosylation profile of ZENAPAX DAC is characterized by peaksrepresenting oligosaccharides G0-GlcNAc, G0, G1, Man5, G2, Man6, Man7and sialylated oligosaccharide. The lower panel of FIG. 21 shows thatthe glycosylation profile of DAC HYP is characterized by two main peakscorresponding to G0-GlcNAc glycoforms and G0 glycoforms and a minor peakcorresponding to a G1 glycoform. The G0-GlcNAc glycoforms can range fromabout 5% to about 20% of the AUC, more typically about 7.2% to 14.6% ofthe AUC. The G0 glycoforms can range from 70% to 99.2% of the AUC, moretypically from 80.9% to 99.2% of the AUC. The G1 glycoform can rangefrom 1% to 9% of the AUC, more typically from 1.4% to 3.8% of the AUC.Sialylated oligosaccharides are 1.0% of the total AUC or less.

Immunogenicity and high levels of effector function can be problematicfor chronically administered drugs. In addition, rapid clearance ratescan reduce drug availability. As is well-known by skilled artisans,differences in glycosylation patterns of therapeutic antibodies can giverise to differences in immunogenicity. Antibodies having highlyhomogeneous glycosylation patterns like DAC HYP may provide beneficialimmunogenicity profiles, ADCC levels, and clearance rates. In addition,biologics having more homogeneous glycosylation patterns reduce batch tobatch variation and can improve consistency and stability.

Accordingly, in another aspect the present disclosure providesdaclizumab compositions that are characterized by a homogeneous N-linkedglycosylation profile. In one embodiment, the daclizumab composition ischaracterized by an N-linked glycosylation profile that includesapproximately 5-20% of the total AUC of G0-GlcNAc glycoforms, and insome embodiments approximately 5%-18% or approximately 7-15% (e.g.,7.2%-14.6% or 6.9%-14.7%) of the total AUC of G0-GlcNAC glycoforms (andin some specific embodiments 7.3% of the total AUC of G0-GlcNAcglycoforms), and approximately 70%-99.2% of the total AUC of G0glycoforms, and in some embodiments approximately 75%-90%, approximately75-92%, or approximately 81-88% of the total AUC of G0 glycoforms (andin some specific embodiments 86% of the total AUC of G0 glycoforms), asmeasured by HPLC. Optionally, the G1 peak is less than about 10% of thetotal AUC, less than about 5%, less than about 4% or less than about 3%of the total AUC and, in certain embodiments, ranges from about 1% toabout 4% (e.g., 1.4% to 3.8%) or about 1% to about 3%. The Man5glycoforms are preferably about 3% of the total AUC or less. In otherembodiments, the daclizumab composition is characterized by an HPLCN-linked glycoform profile substantially similar to a profileillustrated in FIG. 19.

In certain aspects, a daclizumab composition of the disclosure ischaracterized by the sum total of two or more glycoform peaks. Incertain embodiments, the daclizumab compositions of the disclosure arecharacterized by (a) two main peaks corresponding to G0-GlcNAcglycoforms and G0 glycoforms which together range from about 75% toabout 100%, from about 80% to about 100%, or about 85% to about 100% ofthe total AUC and/or (b) peaks corresponding to Man5, Man6, and Man7glycoforms which together are about 6% of the total AUC or less and/or(c) peaks corresponding to Man6 and Man7 glycoforms which together areabout 2% of the total AUC or less. In such embodiments, the percentageof G0-GlcNAc G0, G1, and/or Man5 can be present in the amounts describedin the preceding paragraph.

The binding and inhibitory properties of DAC HYP, as well as thefunctional potency of DAC HYP as evaluated in an assay that measures theinhibition of IL-2-induced proliferation of T-cells, are similar tothose of ZENAPAX DAC. However, quite surprisingly, DAC HYP exhibitssignificantly less ADCC cytotoxicity than ZENAPAX DAC, which is likelydue, at least in part, to differences in their non-fucosylated mannoseglycosylation levels (see FIG. 21). As shown in FIG. 22A and FIG. 22B,DAC HYP exhibits at least 25% less ADCC cytotoxicity than ZENAPAX DAC asmeasured in a cellular assay. As will be recognized by skilled artisans,the reduced ADCC cytotoxicity of DAC HYP may be beneficial forindications involving chronic administration where cell death is notdesirable, for example, for the treatment of multiple sclerosis oruveitis. In these contexts, where therapy is applied chronically, suchas, for example, in the treatment of multiple sclerosis and othernon-oncology indications, DAC HYP therapy may be safer than therapy withZENAPAX™.

Accordingly, in another aspect, the disclosure provides daclizumabcompositions that are characterized by exhibiting ADCC cytotoxicity ofless than about 30%, 25%, 20%, 15%, 10%, 5%, or even lower, at aconcentration of 1 μg/mL as measured in an in vitro assay using aneffector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for examplewhen using Kit225/K6 as a target cell and/or when using PBMC effectorcells from 3 or more, 6 or more, 10 or more, or 50 or more healthydonors. In specific embodiments, the disclosure provides daclizumabcompositions that are characterized by exhibiting ADCC cytotoxicityranging from 5-30%, from 10-30%, from 15-30%, from 15-30%, from 5-25%,from 10-25%, from 20-30%, from 15-25%, from 15-35%, or from 20-35% at aconcentration of 1 μg/mL as measured in an in vitro assay using aneffector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for examplewhen using Kit225/K6 as a target cell and/or when using PBMC effectorcells from 3 or more, 6 or more, 10 or more, or 50 or more healthydonors. The lower levels of ADCC cytotoxicity observed with DAC HYP ascompared to ZENAPAX DAC are surprising given that DAC HYP is an IgG₁immunoglobulin and does not contain framework mutations known to reduceADCC cytotoxicity.

The safety profile of DAC HYP as compared to ZENAPAX DAC may be furtherimproved by the use of a high yield serum free process, that permits theproduction of a highly pure product free of bovine serum albumin (BSA).Accordingly, the present disclosure provides a daclizumab compositionthat is free of BSA and/or is the product of a cell culture process inwhich BSA is not present.

Daclizumab compositions characterized by one or more of the propertiesdiscussed above (DAC HYP compositions) can be conveniently obtained viarecombinant expression in mammalian cells. While not intending to bebound by any particular theory of operation, it is believed that one ormore of the unique characteristics and/or properties discussed above maybe due, at least in part, to the use of a high productivity recombinantexpression system. This can be achieved by any method, such as by geneamplification using the DHFR, or using a selectable marker gene underthe control a weak promoter, preferably in combination with a strongpromoter driving the expression of the protein of interest (preferably asecreted protein). Without being bound by theory, it is believed thatselection of markers under the control of a weak promoter facilitatesthe identification of stable transfectants in which the expressionvector integrates into a chromosomal region that is transcriptionallyactive, yielding high expression levels of the protein of interest. Inone embodiment, the weak promoter driving the expression of a selectablemarker is an SV40 promoter (Reddy et al., 1978, Science 200:494-502) inwhich the activity of one or both enhancer regions has been reduced oreliminated, such as by partial or complete deletion, optionally incombination with a strong promoter, such as the CMV IE promoter (Boshartet al., 1985, Cell 41(2):521-30), driving expression of the protein ofinterest.

Accordingly, in another aspect, the disclosure provides vectors usefulfor generating recombinant cell lines that stably express high levels ofa daclizumab such as DAC HYP, in which expression of the selectionmarker is under the control of an SV40 promoter whose enhancer functionhas been reduced, such as by partial deletion of one or both enhancersequences (designated dE-SV40). A specific dE-SV40 promoter sequencethat can be used to produce stable expression cell lines is at positions6536-6735 of vector pHAT.IgG1.rg.dE (SEQ ID NO:5), illustrated in FIG.3A-FIG. 3D, and in FIG. 3E (SEQ ID NO: 12). Various embodiments ofspecific vectors that can be used to produce stable expression celllines are described in U.S. Application No. 61/565,419 filed Nov. 30,2011 and International Application No. PCT/US11/62720 filed Nov. 30,2011, incorporated herein by reference.

Generally, vectors useful for expressing a daclizumab such as DAC HYPwill include one or more of the features exemplified by pHAT.IgG1.rg.dE(described in Section 7.1 below), such as a promoter. The two chains ofdaclizumab can be placed under separate transcriptional control but arepreferably on the same vector, and their coding regions can be cDNA orgenomic DNA containing introns and exons. As an alternative to separatetranscriptional control, the two chains can be expressed as a singletranscript or a single open reading frame, with their coding regionsseparated by an internal ribosome entry site or a self-cleaving inteinsequence, in which case the heavy and light chain coding sequences areunder the control of a single promoter. An exemplary promoter is the CMVIE promoter and enhancer (at positions 0001-0623 and 3982-4604 ofpHAT.IgG1.rg.dE (SEQ ID NO:5)). Additional features includetranscriptional initiation sites (if absent from the promoter chosen),transcription termination sites, and origins of replication. Examples ofsuch features are illustrated in Table 1, which outlines the componentsof pHAT.IgG1.rg.dE.

A specific embodiment useful for expressing both heavy and light chainsof a daclizumab such as DAC HYP from a single exogenous nucleic acid, inNS0 cells utilizes a selection marker operable in mammalian cells, suchas neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), hygromycin B phosphotransferase (Hph),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), glutaminesynthetase (GS) or Herpes simplex virus thymidine kinase (HSV-tk). In apreferred embodiment, the selectable marker in a vector of thedisclosure is an E. coli guanine phosphoribosyl transferase selectablemarker under the control of an enhancer-less SV40 promoter, the encodingsequence of which can be found at positions 6935-7793 of pHAT.IgG1.rg.dE(SEQ ID NO:5) shown in FIG. 3A-FIG. 3D.

In another aspect, the disclosure provides host cells transfected withvectors useful for recombinantly producing daclizumab, such as forexample, DAC HYP. The host cell may be any mammalian cell, including,for example, Chinese Hamster Ovary (CHO) cells, NS0 murine myelomacells, Sp2/0 cells, PER.C6 cells, Vero cells, BHK cells, HT1080 cells,COS7 cells, WI38 cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2cells, MDCK cells and 293 cells. Once transfected, the vector mayintegrate into the genome to yield a stable production cell line.Skilled artisans will appreciate that it is undesirable to includeanimal products in compositions designated for administration to humans.Accordingly, host cells that do not require serum or other animalproducts for growth (such as, e.g., cholesterol) are preferred. Hostcells that require such animal products can be adapted to utilizeserum-free and other animal product-free medium. A method for adaptingmurine myeloma NS0 cells to grow in serum- and cholesterol-free mediumis described in Hartman et al., 2007, Biotech. & Bioeng. 96(2):294-306and Burky et al., 2007, Biotech. & Bioeng. 96(2):281-293.

As will be recognized by skilled artisans, the basal and feed media usedto culture cells for recombinant protein production, as well as othervariables such as the feeding schedule, growth rate, temperature, andoxygen levels, can affect the yield and quality of the expressedprotein. Methods of optimizing these conditions are within the purviewof a skilled artisan; exemplary conditions are set forth in theExemplary Embodiments of the disclosure. Preferably, cells are adaptedto grow in media free of cholesterol-, serum-, and other animal-sourcedcomponents; in such instances the basal and feed media preferablyinclude defined chemicals that substitute for such components. It hasalso been discovered that media containing high levels of glucose, e.g.,10-35 g/L glucose, advantageously increase the cell cultureproductivity. In a specific embodiment, the basal medium has about 10-20g/L, more preferably about 15 g/L, glucose and/or the feed medium has22-35 g/L, more preferably around 28 g/L, glucose. The feed medium canbe added to the cells according to an escalating feed schedule, as isknown in the art, over a period of 8-15 days, 9-13 days, or, mostpreferably, 10-13 days.

For a DAC HYP expressed in NS0 producer strain 7A11-5H7-14-43, thecomponents of the growth and feed media, and other variables affectingexpression and production have been optimized. Accordingly, thedisclosure also provides optimized basal media, feed media, feedingschedules and other culturing methods and conditions useful forproducing daclizumab in high yield and purity. These media and culturingparameters and methods are described in more detail in Section 7.3.

It has also been discovered that purifying daclizumab from a cellculture utilizing a combination of certain chromatography steps yieldspurified daclizumab and DAC HYP drug substance compositions and liquiddaclizumab and DAC HYP drug formulations that are shelf stable in liquidform at high concentrations, typically at nominal daclizumab or DAC HYPconcentrations of at least about 100 mg/mL+10-15% and in someembodiments 150 mg/mL+10-15% (as measured by UV spectroscopy orrefractive index).

The stable, high concentration daclizumab drug formulations aregenerally prepared by exchanging a concentrated daclizumab formulationwith exchange buffer having an osmolality in the range of about 267-327mOsm/kg (e.g., 270-310 mOsm/kg) and a pH in the range of about pH5.8-6.2 at 25° C. (e.g., 5.9-6.1 at 25° C.) to yield an intermediateformulation, and then diluting the intermediate formulation withpolysorbate dilution buffer to yield a stable, high concentration liquidformulation comprising of about 100 mg/mL+10% daclizumab (e.g., DACHYP), and in some embodiments at least about 150 mg/mL daclizumab (e g.,DAC HYP), as measured by UV spectroscopy or refractive index. Thedilution buffer is the same as the exchange buffer, but includes about0-10% (w/v) polysorbate 80, and is used in an amount such that thefinal, stable, high concentration daclizumab formulation has acalculated polysorbate 80 concentration (nominal concentration) in arange of 0.02-0.04%, in some embodiments about 0.03% (w/v). A variety ofdifferent buffering agents and excipients can be included in theexchange and dilution buffers to achieve an osmolality and pH within thedefined ranges. A specific, non-limiting example of an exchange buffersuitable for formulating stable, high concentration liquid daclizumaband DAC HYP drug formulations contains about 40 mM succinate and about100 mM NaCl and has a pH of about 6.0 at 25° C. A specific, non-limitingexample of a dilution buffer suitable for use with this exchange buffercontains about 40 mM succinate, about 100 mM NaCl and about 1% (w/v)polysorbate 80 and has a pH of about 6.0 at 25° C. The pH of the finalformulation can be adjusted with acid or base to yield an actual pH ofabout 6.0 at 25° C.

The stable, high concentration liquid daclizumab formulations arecharacterized by a low level of aggregation, typically containing atleast 95% monomer and less than 3% aggregates, sometimes less than 1.5%aggregates, and more usually greater than 99% monomer and less than 0.8%aggregates, as measured by size exclusion chromatography. Other puritycharacteristics of the high concentration liquid daclizumab drugformulations are described in more detail in Section 7.6.

The high concentration daclizumab drug formulations are alsocharacterized by a long shelf life, being stable against greater than 5%degradation and formation of greater than 3% aggregates (as measured bySDS-PAGE and size exclusion chromatography, respectively) for periods ofup to 54 months or longer, for example, for at least 5 years, whenstored at 2-8° C., for periods of up to 9 months when stored underaccelerated conditions (23-27° C./60±5% relative humidity) and forperiods of up to 3 months when stored under stressed conditions (38-42°C./75±5% relative humidity).

As noted above, the stable, high concentration liquid daclizumabformulations can be prepared by diluting an intermediate formulationwith polysorbate dilution buffer to yield finished daclizumab drugformulation. Accordingly, in another aspect, the disclosure providespolysorbate-free purified daclizumab (preferably DAC HYP) intermediateformulations containing at least about 150 mg/mL daclizumab, in someembodiments about 170-190 mg/mL daclizumab, that can be diluted withpolysorbate dilution buffer to yield a stable, high concentrationdaclizumab liquid drug formulations as described herein. In a specificembodiment, the concentrated polysorbate-free intermediate formulationsnominally contain about 155 mg/mL or about 180 mg/mL daclizumab(preferably DAC HYP), about 40 mM sodium citrate and about 100 mN NaCl,pH 6.0 at 25° C. In a specific embodiment, the concentratedpolysorbate-free intermediate formulations nominally contain about 155mg/mL or about 180 mg/mL daclizumab (preferably DAC HYP), about 40 mMsodium succinate and about 100 mN NaCl, pH 6.0 at 25° C. The daclizumabcompositions are characterized by a low level of aggregates, describedfurther below.

It has been discovered that concentrating daclizumab via ultrafiltrationinduces aggregates to form, which can result in a high concentrationdaclizumab drug formulation containing unacceptable (e.g., >3%) levelsof aggregates. Accordingly, it is preferable to utilize a “polishing”step prior to concentrating the daclizumab drug substance to removeaggregates. The level of acceptable aggregates prior to concentrationwill depend upon the concentration of the daclizumab drug substance tobe concentrated, the desired concentration in the final daclizumab drugformulation, and the acceptable level of aggregates in the finaldaclizumab drug formulation. For example, if a 150 mg/mL daclizumabformulation containing less than 3% aggregates is desired, and thedaclizumab drug substance must be concentrated 10- to 30-fold (e.g.,20-fold) to achieve this finished daclizumab formulation, the daclizumabcomposition to be concentrated should contain <0.3% aggregates,preferably <0.2% aggregates, and preferably even lower levels, e.g.,about 0.1% aggregates.

A variety of known techniques can be used to obtain a startingdaclizumab drug substance composition containing acceptable levels ofaggregates for concentration into concentrated daclizumab intermediateand final drug formulations as described herein, including, for example,strong cation exchange chromatography and hydrophobic interactionchromatography. However, it has been surprisingly discovered that weakcation exchange chromatography reduces levels of aggregates ofdaclizumab compositions containing in the range of 4-12 mg/mL daclizumaband up to 2.5% aggregates to extremely low levels, typically to about0.1% aggregates. The use of weak cationic exchange to remove aggregatesis more environmentally friendly than hydrophobic interactionchromatography, which utilizes nitrogen containing solutions (such asammonium sulfate solutions).

Accordingly, in another aspect, the disclosure provides methods ofpolishing daclizumab compositions to remove aggregates such that theresulting polished composition generally contains about 4 to 15 mg/mLdaclizumab, where 0.3% or less (e.g., 0.2% or less or 0.1% or less) isin aggregate form, as measured by size exclusion chromatography. Themethod generally involves passing a daclizumab composition containingabout 4-10 mg/mL, typically about 8-9 mg/mL, and preferably about 8.5mg/mL daclizumab and >0.5% aggregates over a weak cation exchange resinin a suitable buffer to adsorb to the daclizumab, and eluting theadsorbed daclizumab with an elution buffer. Useful weak cation exchangeresins include, but are not limited to, CM-650M (Tosoh Biosciences),CM-Sepharose, CM-HyperD. The components of the equilibration, washingand elution buffers will depend upon the weak cation exchange resinused, and will be apparent to those of skill in the art. For CM-650Mresin (Tosoh Biosciences, part Number 101392), an equilibration and washbuffer containing about 20 mM sodium citrate, pH 4.5 and an elutionbuffer containing 20 mM sodium citrate and 75 mM sodium sulfate, pH 4.5works well. The flow rate used will depend upon the choice of resin andsize of the column. For a cylindrical column of CM-650M resin having abed height in the range of about 10-30 cm (e.g., 17-19 cm) and a flowrate in the range of about 50-200 cm/hr (e.g., 90-110 cm/hr, preferablyabout 100 cm/hr), works well with the chromatography can be carried outat room temperature, or at lower temperature, for examples temperaturesranging from 4°, 10°, 15°, 20° or 25° C. A typical useful temperaturerange is 18-25° C. (e.g., 18-22° C.).

According to the ZENAPAX EMEA, the purification process for ZENAPAX DACinvolves the following twelve steps:

-   -   (i) culture broth concentration;    -   (ii) Q-Sepharose chromatography;    -   (iii) S-Sepharose chromatography;    -   (iv) low pH treatment for viral inactivation;    -   (v) concentration/diafiltration;    -   (vi) DV50 filtration for virus removal;    -   (vii) Q-Sepharose II chromatography;    -   (viii) viresolve chromatography for virus removal;    -   (ix) concentration by ultrafiltration;    -   (x) S-300 gel filtration chromatography;    -   (xi) concentration by ultrafiltration;    -   (xii) aseptic filling of vials.

This process is inefficient, and provides a low purification yield. Ithas been discovered that higher yields can be achieved with a processhaving fewer steps, while at the same time yielding a higher degree ofpurity, which permits the resultant daclizumab drug substance to beformulated into high concentration drug formulations as described above.Accordingly, the present disclosure also provides improved methods forisolating and/or purifying both daclizumab drug substance and highconcentration drug formulations. The process utilizes Protein A affinitychromatography in conjunction with strong anion exchange (Q-Sepharose)chromatography and weak cation exchange (CM-650M) chromatography,permitting continuous flow processing without dilution of processintermediate. The improved method for obtaining purified daclizumab drugsubstance involves the following steps:

-   -   (i) protein A affinity chromatography to isolate daclizumab from        other cell culture components;    -   (ii) low pH viral inactivation;    -   (iii) strong anion exchange (Q-Sepharose) chromatography to        remove DNA;    -   (iv) weak cation exchange (CM-650M) chromatography to reduce        aggregates; and    -   (v) filtration to remove viruses.

The exact volumes, column sizes and operating parameters will depend, inpart, on the scale of purification, as is well-known in the art.Specific volumes, column sizes and operating parameters useful forlarge-scale purifications are described in Section 7.4.

Crude daclizumab to be purified and optionally formulated via the abovemethods can be harvested from the cell culture using a variety ofconventional means, e.g., microfiltration, centrifugation, and depthfiltration directly from bioreactor. However, it has been discoveredthat crude daclizumab can be conveniently harvested by lowering the pHof the cell culture to approximately pH 5 at a temperature of less than15° C. to flocculate the cells, which can be removed via centrifugation.In a specific embodiment, crude daclizumab is harvested by lowering thepH of the cell culture to approximately pH 5, chilling the culture toless than 15° C., for example 4° C., for 30-90 minutes, and centrifugingthe resultant suspension to remove cells. This process is generallyapplicable to any cell culture that secretes recombinant proteins intothe culture medium, and is not specific to cultures producing daclizumabor therapeutic antibodies. The pH of the culture can be adjusted using avariety of different acids, including weak or strong organic acids, orweak or strong inorganic acids. For daclizumab cultures, it has beendiscovered that citric acid works well. A concentrated citric acidsolution, e.g., a 0.5 M-2 M solution, can be used for adjusting the pHof the culture prior to harvesting.

The purification of DAC HYP is accomplished by use of threechromatography steps, virus inactivation, virus filtration and finalultra filtrations. Protein A affinity chromatography is the first stepin the purification process, which clears the majority of processrelated impurities. To enable the reuse of protein A affinity column, itmust be regenerated and sanitized. It has been discovered that aqueousNaOH solution is effective in accomplishing both column regeneration andsanitization. However, the use of NaOH solutions can degrade the proteinA resin, increasing overall production costs. It has also beendiscovered that sanitizing protein A affinity chromatography resins witha solution containing NaOH and benzyl alcohol yields good results andsignificantly increases the number of purification cycles. Accordingly,the disclosure also provides a sanitization solution and method forregenerating and sanitizing protein A affinity columns and resins. Thebuffer generally comprises about 100 to 500 mM sodium citrate, about 10to 30 mM NaOH and about 0.5 to 3% (v/v) of benzyl alcohol, and has a pHin the range of about pH 10 to 13. The buffer may also optionallyinclude other components, such as, for example, salts and/or detergents.Both sodium citrate and benzyl alcohol are important for protectingprotein A resin from being destroyed by NaOH and enhancing microbicidalactivities. In specific embodiments, the Protein A sanitization buffercontains about 200 mM sodium citrate, about 20 mM NaOH, and about 1%(v/v) benzyl alcohol. As described in Section 7.4.2, sanitizationsolutions containing benzyl alcohol and sodium hydroxide have beneficialantimicrobial effects, and can be used to sanitize protein A columns inpurification processes for any antibody.

The sanitization buffer can be used to sanitize Protein A chromatographyresin in a batch-wise process, where the resin is washed with excess(e.g., 1.5-2× volumes) of sanitization buffer followed by incubation forabout 30-45 min. in excess (e.g., 1.5-2× volumes) sanitization buffer,followed by equilibration with equilibration buffer or storage buffer.The sanitization buffer can also be used to sanitize a prepared ProteinA chromatography column by washing the column with excess (e.g., 1.5-2×column volumes) sanitization buffer at a suitable flow rate (e.g.,ranging from about 110-190 cm/hr, or 135-165 cm/hr), holding the columnunder conditions of zero flow for about 30-40 min, and then washing thecolumn with equilibration buffer or storage buffer. Suitableequilibration and storage buffers are described in Section 0.

Sanitizing Protein A columns with the sanitization buffers describedherein significantly increases the number of purifications for which asingle batch of resin can be used. For example, whereas a single batchof Protein A resin typically lasts only about 30 purification cycleswhen sanitized with conventional NaOH buffers (e.g., 50 mM NaOH, 0.5 MNaCl), Protein A columns sanitized with the sanitization buffersdescribed herein can be used for more than 100 purification cycles.While not intending to be bound by any theory of operation, it isbelieved that the sanitization buffers described herein in part protectthe immobilized Protein A from NaOH-induced degradation, therebyincreasing the useful life of the resin. Accordingly, while improvementsare expected for all Protein A resins, including those that utilizemutant strains of Protein A designed to be resistant to NaOH degradation(for example MabSuRe resin), the sanitization buffers described hereinare especially beneficial when used to sanitize Protein A resins andcolumns utilizing unmodified immobilized Protein As, or Protein As thathave not been engineered to be NaOH stable. The disclosure furtherprovides methods comprising using a protein A affinity resin for morethan 30, more than 35 or more than 40 antibody purification runs, and insome instances up to 50 or up to 100 protein purification cycles,comprising conducting the purification runs and washing the resin with asanitization solution as disclosed herein.

As mentioned above, daclizumab specifically binds CD25 expressed onactivated and not resting T and B lymphocytes, and blocks binding ofIL-2 to CD25, thereby inhibiting formation of the high affinity IL-2receptor complex, inhibiting proliferation of the activated T- andB-cells. The DAC compositions and formulations described herein, and inparticular the DAC HYP compositions and formulations, likewisespecifically bind CD25 and exhibit similar biological properties. TheDAC compositions and formulations described herein, and in particularDAC HYP, are therefore useful in any of the assays and therapeuticmethods described for daclizumab generally, and ZENAPAX specifically.Accordingly, the present disclosure also provides methods of using theDAC compositions and formulations described herein, and in particularthe DAC HYP compositions and high concentration stable liquidformulation, to inhibit proliferation of activated T- and B-cells, bothin in vitro applications and in vivo as a therapeutic approach towardsthe treatment of diseases in which activated T- and/or B-cellproliferation play a role, such as the treatment and prevention ofallograft rejection, the treatment of uveitis, and the treatment ofmultiple sclerosis.

The methods generally involve contacting an activated T- and/or B-cellwith an amount of a daclizumab composition or formulation describedherein sufficient to inhibit its proliferation.

For methods of treatment, the methods generally involve administering toa subject an amount of a daclizumab composition, for example a DAC HYPcomposition or a high concentration DAC formulation as described herein,to provide therapeutic benefit. In a specific embodiment, the daclizumabcompositions and formulations can be used to treat multiple sclerosis,either alone or in combination with other agents such as interferonbeta. The DAC compositions described herein can be administeredsubcutaneously to a patient from weekly to monthly (e.g., weekly, everytwo weeks, twice a month, every four weeks or monthly) in doses rangingfrom 75 mg to 300 mg (e.g., 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200mg, 225 mg, 250 mg, 275 mg or 300 mg) or ranging from 1 mg/kg to 4mg/kg. The compositions can be provided in prefilled syringes convenientfor subcutaneous use, preferably at nominal daclizumab concentrations of100 mg/mL+10-15% or 150 mg/mL+10-15%. The concentrated DAC compositionscan also be diluted for intravenous administration.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides DAC-HYP light chain cDNA (SEQ ID NO: 1) and translatedamino acid (SEQ ID NO:2) sequences. The bold, underlined aspartate (D)residue is the first amino acid in the properly processed matureprotein; the amino acid sequence upstream of this residue corresponds tothe signal sequence.

FIG. 2 provides DAC-HYP heavy chain cDNA (SEQ ID NO:3) and translatedamino acid sequences (SEQ ID NO:4). The bold, underlined glutamaine (Q)residue is the first amino acid in the properly processed matureprotein; the amino acid sequence upstream of this residue corresponds tothe signal sequence.

FIG. 3A-FIG. 3D together provide the full nucleotide sequence for vectorpHAT.IgG1.rg.dE (SEQ ID NO:5).

FIG. 3E provides a specific embodiment of a dESV40 promoter (SEQ IDNO:12) that can be used to select high yielding producer strains.

FIG. 4A-4B provide a schematic diagram of vector pHAT.IgG1.rg.dE (FIG.4A), which is derived from pABX.gpt, a vector that can be adapted toexpress any heavy and light chain genes or even a non-antibodypolypeptide (FIG. 4B).

FIG. 5 provides an exemplary production process for DAC HYP.

FIG. 6 demonstrates UV (280 nm), pH and conductivity monitoring ofproduct fractions during protein A affinity chromatography.

FIG. 7 demonstrates UV (280 nm), pH and conductivity monitoring ofproduct fractions during Q-sepharose chromatography.

FIG. 8 demonstrates UV (280 nm), pH and conductivity monitoring ofproduct fractions CM cation exchange chromatography.

FIG. 9 provides a schematic illustration of the DAC HYP ultrafiltrationsystem.

FIG. 10 provides a 0-60 minute DAC HYP peptide map chromatogram. Thereference profile is a 100 mg/ml DAC HYP preparation and Batch 1 andBatch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 11 provides a 55-115 minute DAC HYP peptide map chromatogram. Thereference profile is a 100 mg/ml DAC HYP preparation and Batch 1 andBatch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 12 provides a 110-170 minute DAC HYP peptide map chromatogram. Thereference profile is a 100 mg/ml DAC HYP preparation and Batch 1 andBatch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 13 provides overlaid circular dichroism spectra of DAC HYP 150mg/ml lots Batch 1 and Batch 2. The reference is a 100 mg/ml preparationof DAC HYP.

FIG. 14A-FIG. 14B provides overlaid zero-order ultraviolet spectra andoverlaid second derivative ultraviolet spectra, respectively. Thereference profile is a 100 mg/ml DAC HYP preparation and Batch 1 andBatch 2 correspond to 150 mg/ml DAC HYP preparations. All three spectraare present in each of FIG. 14A and FIG. 14B, but appear as a singlespectrum once overlaid on one another.

FIG. 15A-FIG. 15B provide full scale and expanded scale size exclusionchromatograms, respectively. The reference profile is a 100 mg/ml DACHYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYPpreparations.

FIG. 16 is a plot of DAC HYP aggregation as a function of time.

FIG. 17 shows reduced and non-reduced SDS-PAGE (left and right panels,respectively). The reference profile is a 100 mg/ml DAC HYP preparationand Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 18 shows cation exchange chromatograms of DAC HYP. The referenceprofile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2correspond to 150 mg/ml DAC HYP preparations. The peak labels correspondto the different N- and C-terminal isoforms.

FIG. 19 shows HPLC chromatograms of N-linked oligosaccharidesenzymatically cleaved from DAC HYP. The reference profile is a 100 mg/mlDAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DACHYP preparations.

FIG. 20 shows ADCC response curves of DAC HYP. The reference profile isa 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to150 mg/ml DAC HYP preparations.

FIG. 21 shows HPLC chromatograms for N-linked oligosaccharides releasedfrom DAC HYP (lower panel) and ZENAPAX DAC (upper panel) illustratingtheir different glycosylation profiles.

FIG. 22A-FIG. 22B provide a comparison between the ADCC activity of twoDAC HYP preparations (referred to as DAC HYP Batch 3 and DAC HYP Batch4), DAC Penzburg, and ZENAPAX DAC using the variable effector-to-targetcell ratio ADCC assay format (FIG. 22A) and the variable antibodyconcentration ADCC assay format (FIG. 22B).

FIG. 23 provides a comparison of the charge isoforms of DAC HYP, DACPenzberg and ZENAPAX DAC.

6. DETAILED DESCRIPTION

The present disclosure provides, among other things, DAC compositionshaving specified properties, high concentration DAC formulationsespecially useful for certain modes of administration that are shelfstable at different temperatures, vectors and host cells useful forproducing the DAC compositions, optimized culture broths and culturingconditions useful for producing the DAC compositions, methods forpurifying the DAC compositions and high concentration formulations, andmethods of using the DAC compositions and high concentrationformulations, for example to inhibit proliferation of activated T-and/or B-cells and to treat and/or prevent activated T- and/or B-cellmediated diseases, such as, for example, multiple sclerosis.

Daclizumab (DAC) as used herein refers to a humanized IgG₁ monoclonalantibody having the light (V_(L)) chain sequence illustrated in FIG. 1(positions 21-233 of SEQ ID NO:2) and the heavy (V_(H)) chain sequenceillustrated in FIG. 2 (positions 20 to 465 of SEQ ID NO:4). The CDRsequences of DAC are as follows:

V_(L)CDR#1:  (SEQ ID NO: 6) SASSSISYMH  V_(L)CDR#2:  (SEQ ID NO: 7)TTSNLAS V_(L)CDR#3:  (SEQ ID NO: 8) HQRSTYPLT V_(H)CDR#1: (SEQ ID NO: 9) SYRMH V_(H)CDR#2:  (SEQ ID NO: 10) YINPSTGYTEYNQKFKD V_(H)CDR#3:  (SEQ ID NO: 11) GGGVFDY

Certain daclizumab molecules have been reported in the literature, and aspecific version of DAC has been previously marketed under the tradenameZENAPAX by Hoffman-La Roche for the prevention of allograft rejection inrenal transplant patients as an adjunct to immunotherapy includingcyclosporin and corticosteroids. The version of DAC sold under thetradename ZENAPAX is referred to herein as “ZENAPAX DAC.”

Another version of DAC, produced at a facility in Penzberg, Germany,although never sold commercially, has been used in certain clinicaltrials. This version of DAC is referred to herein as “DAC Penzberg.”

As described herein, the present disclosure concerns, in part, a newversion of DAC having characteristics and properties that differ from,and in some instances that are superior to, the characteristics andproperties of ZENAPAX DAC and DAC Penzberg. Accordingly, the presentdisclosure in part concerns DAC compositions that are new. The new DACcompositions are characterized by one or more of the following features,as described more fully in the Summary section:

(1) Characteristic pE/Q and/or Q/VHS N-terminal isoforms;

(2) A homogeneous N-linked oligosaccharide profile characterized by twomain peaks and a minor peak;

(3) Reduced ADCC cytotoxicity as compared to ZENAPAX DAC and DACPenzberg; and

(4) A low level of aggregate forms (<3%) when formulated at nominalconcentrations as high as 150±10-15%.

DAC compositions having one or more of these characteristics and/orproperties are referred to herein as “DAC HYP” compositions. Forpurposes of exemplifying the various aspects and features of inventionsdescribed herein, a specific DAC HYP having all four of the aboveproperties is described, as are specific compositions and methods forits production and purification. However, it is to be understood that aDAC HYP composition need not have all of the above four characteristicsto fall within the scope of the disclosure. In specific embodiments, DACHYP has at least two of characteristics (1) through (4) above (e.g., atleast a combination of (1) and (2); (1) and (3); (1) and (4); (2) and(3); (2) and (4); or (3) and (4)) or at least three of characteristics(1) through (4) above (e.g., at least a combination of (1), (2) and (3);(1), (2) and (4); (1), (3), and (4); and (2), (3), (4)). Such DAC HYPcompositions can also have <3% aggregates, <2% aggregates and even lowerlevels, e.g., <1% aggregates, when formulated at concentrations of 100mg±10-15% or even 150±10-15%.

Moreover, while certain aspects and embodiments of the inventionsdescribed herein are illustrated and exemplified with DAC HYP, skilledartisans will appreciate that they are not limited to DAC HYP, and areuseful for daclizumab compositions generally, and also to IgG₂, IgG₃,and IgG₄ anti-CD25 antibodies having specific CD25 binding propertiessimilar to DAC, and to anti-CD25 antibodies suitable for administrationto humans that have not been humanized. These various differentanti-CD25 antibodies are referred to herein as “DAC analogs.” Such DACanalogs may usually include the six DAC CDRs mentioned above, but mayinclude other CDR sequences.

The characteristics and properties of DAC HYP compositions can beconfirmed using standard assays and methods. For example, N-terminal andC-terminal isoform profiles can be assessed using cation exchangechromatography with detection at 220 nm. In a specific method, 100 μL oftest sample (1 mg/mL antibody dissolved in Buffer A) is resolved at roomtemperature on a ProPac WCX-10 column (Dionex Coporation) equipped witha ProPac WCX-10G guard column (Dionex Corporation) using the followingseparation gradient (column is equilibrated with Buffer A):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0.0 100 0 1 60.0 4060 1 80.0 0 100 1 85.0 0 100 1 85.1 100 0 1 100.0 100 0 1 Buffer A = 15mM sodium phosphate, pH 5.9 Buffer B = 250 mM NaCl, 15 mM sodiumphosphate, pH 5

N-linked glycosylation profiles can be assessed by cleaving the N-linkedoligosaccharides with amidase PNGase F, derivatizing theoligosaccharides with a fluorescent label and analyzing the resultantmixture via normal phase HPLC with fluorescent detection. In a specificmethod, anthranilic acid-derivatized, cleaved N-linked glycans areresolved at 50° C. on a 250×4.6 mm polymeric-amine bonded Asahipak AminoNH₂P-504E column (5 m particle size, Phenomenex, cat. No. CHO-2628)using the following elution gradient (using a sample injection volume of100 μL; column is equilibrated with 85% Buffer A/15% Buffer B):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0 85 15 1 2 85 15 110 80 20 1 60 55 45 1 70 5 95 1 75 5 95 1 76 85 15 1 90 85 15 1 Buffer A= 1% v/v tetrahydrofuran, 2% v/v acetic acid in acetonitrile Buffer B =1% v/v tetrahydrofuran, 5% v/v acetic acid, 3% v/v triethylamine inwater

Purity can be confirmed using reduced SDS-PAGE (Precast 14% Tris-Glycinegradient minigels, Invitrogen Part No. 601632) and colloidal bluestaining, and/or size exclusion chromatography with detection at 280 nm.In particular, 15 μL test sample (20 mg/mL antibody in elution buffer)can be resolved at room temperature on a 7.8 mm×30 cm TSK G3000SWXLcolumn (Tosoh Biosciences, part no. 601342) equipped with a 0.5 μmpre-column filter (Upchurch, part no. A-102X) using an isocraticgradient of elution buffer (200 mM KPO4, 150 mM KCl, pH 6.9) at a flowrate of 1 mL/min.

The DAC HYP compositions and other DAC formulations described herein,such as the stable, high concentration liquid DAC formulations describedherein, are useful for treating a variety of disorders and conditionsthought to be mediated, at least in part, by activated T- and/orB-cells, including, for example, rejection of allograft transplants andmultiple sclerosis. Specific patient populations, formulations, modes ofadministration and dosage amounts and schedules useful for treating orpreventing allograft rejection are described in U.S. Pat. No. 6,013,256,and are incorporated herein by reference. Specific patient populations,formulations, modes of administration, dosage amounts and schedulesuseful for treating patients with multiple sclerosis are described inU.S. Pat. No. 7,258,859, and are incorporated herein by reference. Allof these formulations, modes of administration, dosing amounts andschedules, as well as disclosed specific patient populations andcombination therapies, are equally suited to the DAC HYP compositionsand, where applicable, the high concentration DAC formulations,described herein.

The DAC HYP compositions and formulations described herein areadministered in amounts that provide therapeutic benefit. Therapeuticbenefit includes, but is not limited, treatment of the underlyingdisorder. Therapeutic benefit may also include improving or amelioratingsymptoms or side effects of a particular disease as assessed usingstandard diagnostic and other tests. For multiple sclerosis, variousmeans of assessing therapeutic benefit, including, for example, the useof magnetic resonance imaging to assess brain lesions and/or assessingprogression to disability are described in U.S. Pat. No. 7,258,859,incorporated herein by reference. All of these various tests can be usedto assess therapeutic benefit in the context of patients suffering frommultiple sclerosis.

The stable high concentration DAC formulations, whether made with DACHYP, DAC generally or a DAC analog, are particularly useful forsubcutaneous administration in the treatment of chronic diseases such asmultiple sclerosis. The formulations can conveniently be administered asa single bolus subcutaneous injection or diluted for intravenousadministration. The formulations can be administered subcutaneously to apatient from weekly to monthly (e.g., weekly, every two weeks, twice amonth, every four weeks or monthly) in doses ranging from 75 mg to 300mg (e.g., 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg,275 mg or 300 mg) or ranging from 1 mg/kg to 4 mg/kg. The compositionscan be provided in prefilled syringes convenient for subcutaneous use.The diluted formulations can be administered intravenously at suitabledosages at the same frequencies as for subcutaneous administration.

7. EXEMPLARY EMBODIMENTS

Various aspects and features of the inventions described herein aredescribed further by way of the exemplary embodiments, below. It will beappreciated that while the exemplary embodiments utilize specific cellculture media, cell culture conditions, column chromatography resins andequilibration, washing and elution buffers, routine changes can be made.Moreover, while the various cell culturing methods are exemplified witha specific producer strain (clone 7A11-5H7-14-43, also referred to asDaclizumab dWCB IP072911), it is expected that other DAC or DAC analogproducer strains could be used with success, with or without routineoptimization. Moreover, features that are described in association witha particular embodiment (whether in the Summary above or in theExemplary Embodiments that follow) can be deviated from withoutsubstantially affecting the desirable properties of the methods andcompositions of the disclosure, and moreover that different embodimentscan be combined and used in various ways together unless they areclearly mutually exclusive. Accordingly, it is to be understood that theexemplary embodiments provided below are intended to be illustrative andnot limiting, and should not be construed as limiting the claims thatfollow these embodiments.

The manufacturing method exemplified below was used to produce a DAC HYPdrug substance at 150 mg/mL. For making a DAC HYP drug substance at 100mg/mL, small process changes are introduced:

The cell culture used to produce a DAC HYP (see Section 7.3) at 100mg/mL does not include an antifoam emulsion, whereas the cell cultureDAC HYP at 150 mg/mL uses a low concentration Dow Corning Antifoam C inthe 10,000 L bioreactor to minimize foaming. The CM-650M column (seeSection 7.4.5) is sanitized with a buffer of 0.5 M NaOH, 0.5 M sodiumsulfate when producing a DAC HYP formulation having a final antibodyconcentration of 100 mg/mL; the sodium sulfate is omitted from thesanitization buffer when producing a DAC HYP formulation having a finalantibody concentration of 150 mg/mL. For making DAC HYP at 100 mg/mL, aone step ultrafiltration/diafiltration (UF/DF) is used at the end of thedownstream process immediately prior to addition of polysorbate 80 anddilution of the drug substance to final volume (see Section 7.4.7),whereas for making the drug substance at a concentration to 150 mg/mL, atwo step UF/DF is used. The examples below show comparative analysesamong various lots of DAC HYP at 100 mg/mL and at 150 mg/mL. In severalstudies, batch of DAC HYP at 150 mg/mL were compared against a lot ofDAC HYP 100 mg/mL manufactured at the 10,000 L scale, referred to belowas Reference Standard lot RS0801.

7.1. DAC HYP Expression Construct

The hybridoma producing anti-Tac, a murine IgG_(2a) monoclonal antibody,was generated by fusing the murine myeloma cell line NS-1 withspleenocytes from a mouse immunized with a human T-cell line developedfrom a T-cell leukemia patient (Uchiyama et al., 1981, J. Immunol.126(4):1393-7). Anti-Tac was selected for its reactivity with activatedT-cells, but not with resting T-cells or B-cells. Anti-Tac was latershown to react with the alpha subunit of human IL-2 receptor (Leonard etal., 1982, Nature 300(5889):267-9).

The amino acid sequences for the light and heavy chain variable regionsof the murine anti-Tac were determined from the respective cDNA (Queenet al., 1989, Proc. Nat'l Acad. Sci. USA 86(24):10029-33). The bindingaffinity of the mouse anti-Tac was retained in the humanized form asdescribed in Queen et al. The complementarity determining regions (CDRs)of the murine anti-Tac were first grafted onto the acceptor framework ofhuman antibody Eu. With the aid of a three-dimensional model, key mouseframework residues critical for the conformation of the CDRs and thusthe binding affinity were identified and substituted for the humancounterpart in the acceptor frameworks. In addition, atypical aminoacids in the acceptor frameworks were replaced with the human consensusresidues of the corresponding positions to eliminate potentialimmunogenicity.

DAC HYP V_(L) and V_(H) genes were constructed as mini-exons byannealing and extension of overlapping oligonucleotides as described inQueen et al. (1989). For expression of DAC HYP in the IgG₁ form, theresultant V_(L) and V_(H) genes were cloned into a single expressionvector, as outlined in Cole et al. (1997, J. Immunol. 159(7):3613-21)and Kostelny et al. (2001, Int. J. Cancer 93(4):556-65), to constructpHAT.IgG1.rg.dE (see FIG. 3 and FIG. 4A). Plasmid pHAT.IgG1.rg.dEcontains the genes for both the IgG₁ heavy and kappa light chains ofdaclizumab, each under control of the human cytomegalovirus (CMV)promoter. The plasmid contains the E. coli guanine phosphoribosyltransferase (gpt) gene as a selectable marker. The genetic components inpHAT.IgG1.rg.dE are described in Table 1 below.

TABLE 1 Genetic Components of pHAT.IgG1.rg.dE Restriction NucleotideNumber Sites in Vector Description Reference 0001-0623 EcoRI-XbaI CMV IEenhancer and promoter Boshart et al., 1985, Cell 41(2): 521-30 0624-1056XbaI-XbaI DAC HYP V_(H) 1057-3852 XbaI-BamHI Human Cγ1 Ellison et al.,1982, Nucleic Acids Res. 10(13): 4071-79 3853-3981 BamHI-EcoRITranscription termination site from human Ashfield et al., 1991, EMBO J.10(13): 4197-207 complement gene C2 3982-4604 EcoRI-XbaI CMV IE enhancerand promoter Boshart et al., 1985, Cell 41(2): 521-30 4605-5001XbaI-XbaI DAC HYP V_(L) 5002-6524 XbaI-BamHI Human CK Hieter et al.,1980, Cell 22(1 Pt 1): 197-207. 6525-6735 BamHI-HindIII SV40 enhancerand promoter Reddy et al., 1978, Science 200: 494-502 6736-7793HindIII-Sau3AI E. coli gpt gene Richardson et al., 1983, Nucleic AcidsRes. 11(24): 8809-16 7794-8403 Sau3AI-Sau3AI SV40 intron Reddy et al.,1978, Science 200: 494-502 8404-8639 Sau3AI-BamHI SV40 poly A Reddy etal., 1978, Science 200: 494-502  8640-10936 BamHI-EcoRI pBR322 regionincluding amp gene Sutcliffe, 1979, Cold Spring Harb Symp Quant Biol. 43Pt 1: 77-90

The dESV40 promoter spans positions of 6536-6735 of pHAT.IgG1.rg.dE(6536-6562 is 27 residues of 72 bp enhancer A; 6566-6629 are the three21-bp repeats. 6536-6735 are the reverse complement of 5172-1 and 1-133in GenBank: J02400.1 (Simian Virus 40 Complete Genome)). The nucleotidesequences of DAC HYP light and heavy chain genes in the expressionvector were confirmed by DNA sequencing.

7.2. DAC HYP Stable Cell Line

Mouse myeloma cell line NS0 was obtained from European Collection ofCell Cultures (ECACC catalog #85110503, Salisbury, Wiltshire, UK). Avial of these NS0 cells was thawed into DMEM supplemented with 10% FBS.Cells were maintained in a humidified incubator at 37° C. and 7.5% CO₂.The cells were subsequently cultured in basal medium SFM-3 supplementedwith 1 mg/mL BSA. SFM-3 is a 1:1 mixture of DMEM and Ham's F-12supplemented with 10 mg/mL insulin and 10 μg/mL transferrin. Over aperiod of approximately 3 months, the NS0 cells were adapted to SFM-3without supplements, by gradually reducing the amount of FBS present inthe culture medium until it was eliminated, and then finally removingBSA in a single step. The resulting host cell line was passaged 15-20times in SFM-3 and a frozen bank was prepared.

The SFM-3 adapted cells were transfected with pHAT.IgG1.rg.dE(linearized with FspI enzyme (New England Biolabs, cat. no. R0135L, lot43)) by electroporation. Briefly, 30-40 μg of pHAT.IgG1.rg.dE was addedto 1×10⁷ exponentially growing adapted NS0 cells and pulsed twice at 1.5kV, 25 μF using a Gene Pulser instrument (BioRad, Richmond, Calif.).Following electroporation, cells were plated in DMEM±10% FBS in five96-well plates at 20,000 cells/well, a density that favored a singlecolony per well after mycophenolic acid (“MPA”) selection. As describedin Hartman et al., 2007, Biotech. & Bioeng 96(2):294-306, transfectantsthat had stably integrated the vector were selected in the presence ofmycophenolic acid. Starting from an NS0 stable transfectant thatproduced a high level of DAC HYP, three successive rounds of subcloningwere performed by either limited dilution cloning or fluorescenceactivated cell sorting (FACS) into PFBM-1 containing either 2.5% or 5%fetal bovine serum (FBS; HyClone, Logan, Utah). At each round ofsubcloning, one of the best producers was used for the next round ofsubcloning. Following the third round of subcloning, the finalproduction cell line (7A11-5H7-14-43, also referred to as DaclizumabdWCB IP072911) was chosen. A seed bank of the final production cell linewas then prepared by freezing 1×10⁷ cells per vial in 1 mL of 90%FBS/10% DMSO (Sigma, St. Louis, Mo.).

7.3. DAC HYP Recombinant Production

7.3.1. Cell Culture and Recovery

Cells are thawed from a single cell bank vial and expanded inprogressively larger volumes within T-flasks, roller bottles, spinnerflasks, and bioreactors until the production scale is achieved. Uponcompletion of the production culture, the cell culture fluid isclarified by centrifugation and depth filtration, and transferred to aharvest hold tank. The production culture duration is approximately 10days.

Cell culture and recovery can be carried out in a variety of differentcell culture facilities using standard equipment, as is known in theart. In another example, cells are thawed from a single cell bank vialand expanded in progressively larger volumes within shaker flasks andbioreactors until the production scale is achieved. Upon completion ofthe production culture, the cell culture fluid is clarified bycentrifugation and depth filtration, and transferred to a harvest holdtank. The production culture duration is approximately 10 days.

7.3.1.1. Inoculum Preparation

Production batches are initiated by thawing a single cell bank vial.Cells are transferred to a T-flask containing a chemically-definedmedium, Protein Free Basal Medium-2 (PFBM-2). Custom Powder for makingPFBM-2 can be ordered from Invitrogen by requesting Hybridoma-SFM mediapowder prepared without NaCl, phenol red, transferrin, and insulin,including a quantity of EDTA iron (III) sodium salt that, whenreconstituted, yields a concentration of 5 mg/L, and that has quantitiesof the remaining components adjusted such that, when reconstituted,their concentrations are the same as reconstituted Hybridoma-SFM.Prepared PFBM-2 medium contains the following components: 8 g/L CustomPowder; 2.45 g/L sodium bicarbonate; 3.15 g/L NaCl; and 16.5 g/LD-glucose monohydrate (15 g/L glucose).

The cells are expanded by serial passage into roller bottles or spinnerflasks every two days thereafter. T-flasks, roller bottles, and spinnerflasks are placed in an incubator operating under a temperature setpoint of 37° C. under an atmosphere of 7.5% CO₂ for T-flasks and rollerbottles and 5% CO₂ for spinner flasks.

The spinner flasks are supplemented with 5% CO₂ either by overlay intothe headspace or by sparge into the culture, depending on the cellculture volume, and impeller speed is controlled at constant revolutionsper minute (RPM). The target seeding density at all inoculum expansionpassages is approximately 2.5×10⁵ viable cells/mL.

Furthermore, inoculum preparation can be carried out according tomethods known in the art, using a variety of standard culture vessels,volumes, and conditions. For example, production batches can beinitiated by thawing a single cell bank vial. Cells can be transferredto a shaker flask containing a chemically-defined medium, Protein FreeBasal Medium-2 (PFBM-2). Custom Powder for making PFBM-2 can be orderedfrom Invitrogen by requesting Hybridoma-SFM media powder preparedwithout NaCl, phenol red, transferrin, and insulin, including a quantityof EDTA iron (III) sodium salt that, when reconstituted, yields aconcentration of 5 mg/L, and that has quantities of the remainingcomponents adjusted such that, when reconstituted, their concentrationsare the same as reconstituted Hybridoma-SFM. Prepared PFBM-2 mediumcontains the following components: 8 g/L Custom Powder; 2.45 g/L sodiumbicarbonate; 3.15 g/L NaCl; and 16.5 g/L D-glucose monohydrate (15 g/Lglucose). Optionally, at the bioreactor stage, cupric sulfateheptahydrate can be added, e.g., at a concentration of 0.04 mg/L.

The cells are expanded by serial passage into shaker flasks every twodays thereafter. Shaker flasks are placed in an incubator operatingunder a temperature set point of 37° C. under an atmosphere of 7.5% CO₂.

The shaker flasks are agitated at constant revolutions per minute (RPM)on a shaker platform in the incubators. The target seeding density atall inoculum expansion passages is approximately 2.2-2.5×10⁵ viablecells/mL.

Approximately 14 days following cell bank thaw, when a sufficient numberof viable cells have been produced, the first of several, typicallythree or four, stainless steel stirred-tank seed bioreactors isinoculated. Prior to use, the seed bioreactors are cleaned-in-place,steamed-in-place, and loaded with the appropriate volume of PFBM-2culture medium. The pH and dissolved oxygen probes are calibrated priorto the bioreactor being steamed-in-place. The first seed bioreactor isinoculated with a sufficient number of cells to target an initial celldensity of 2.0-2.5×10⁵ viable cells/mL. Sequential transfer to thelarger volume (typically, 100 L to 300 L and then to the 1,000 L seedbioreactors, or 60 L to 235 L, 950 L, and 3750 L seed bioreactors) isperformed following approximately two days of growth in each reactor andtarget initial cell densities of 2.0-2.5×10⁵ viable cells/mL. Culture pHis maintained by addition of either CO₂ gas or 1 M sodium carbonate(Na₂CO₃) via automatic control. The target operating conditions of theseed and production bioreactors include a temperature set point of 37°C., pH 7.0 and 30% dissolved oxygen (as a percentage of air saturation).The 100 L, 300 L and 1,000 L bioreactors are agitated at 100 rpm, 80 rpmand 70 rpm, respectively. In some instances, the target operatingconditions of the seed and production bioreactors include a temperatureset point of 37° C., a pH of 7.0 with CO₂ sparge and base additioncontrol and 30% dissolved oxygen (as a percentage of air saturation).The larger volume bioreactors can be agitated at speeds of 100 rpm, 80rpm, 70 rpm, or 40 rpm.

7.3.2. Cell Culture Production Bioreactor

After approximately 2 days in the 1,000 L seed bioreactor, the inoculumis transferred into a stainless steel stirred-tank productionbioreactor. The production bioreactor has a working volume ofapproximately 10,000 L. Prior to use, the bioreactor iscleaned-in-place, steamed-in-place, and loaded with approximately 4,000L of PFBM-2 medium. The pH and dissolved oxygen probes are calibratedprior to the bioreactor being steamed-in-place.

In another example, the inoculum is grown in a 3750 L seed bioreactorbefore transfer to a stainless steel stirred-tank production bioreactorwith a working volume of approximately 15,000 L, which iscleaned-in-place, steamed-in-place, and loaded with approximately4,000-7,000 L of PFBM-2 medium prior to use.

The target seeding density of the production bioreactor is in the rangeof 2.0-2.5×10⁵ viable cells/mL. A chemically-defined Protein Free FeedMedium concentrate (PFFM-3) (a chemically-defined concentrated feedmedium made by reconstituting PFFM3 subcomponents 1 and 2, L-glutamine,D-glucose, sodium phosphate dibasic heptahydrate, L-tyrosine, folicacid, hydrochloric acid, and sodium hydroxide) is added during culture.PFFM3 contains the components shown in Table 4:

TABLE 4 PFFM3 Medium Components Component Concentration PFFM3Subcomponent 1 (amino acids) 20.4 g/L prepared PFFM3 Subcomponent 2(vitamins and 4.93 g/L prepared trace elements) L-Glutamine 11.0 g/Lprepared D-Glucose 28.0 g/L prepared L-Tyrosine, disodium salt 1.32 g/Lprepared Folic Acid 0.083 g/L prepared Na₂HPO₄•7H₂0 1.74 g/L preparedSodium Hydroxide Varies, pH control Glacial Hydrochloric Acid Varies, pHcontrol WFI water

PFFM3 Subcomponent 1 contains the components shown in Table 5 below:

TABLE 5 PFFM3, Subcomponent 1 Medium Components MW (g/mole) Conc. (mg/L)Conc. (mM) L-Arginine HCl 211. 1,900 9.00E+00 L-Asparagine Anhydrous132.1 1,320 9.99E+00 L-Aspartic Acid 133.1 119 8.94E−01 L-CysteineHCl•H₂O 176.0 2,030 1.15E+01 L-Glutamic Acid 147.1 510 3.47E+00 Glycine75.1 157 2.09E+00 L-Histidine HCl•H₂O 210.0 864 4.11E+00 L-Isoleucine131.2 1,440 1.10E+01 L-Leucine 131.2 3,130 2.39E+01 L-Lysine HCl 183.02,160 1.18E+01 L-Methionine 149.2 1,260 8.45E+00 L-Phenylalanine 165.2918 5.56E+00 L-Proline 115.1 806 7.00E+00 L-Serine 105.1 709 6.75E+00L-Threonine 119.1 1,220 1.02E+01 L-Tryptophan 204.2 408 2.00E+00L-Valine 117.1 1,450 1.24E+01

PFFM3 Subcomponent 2 contains components shown in Table 6 below:

TABLE 6 PFFM3, Subcomponent 2 Medium Components MW g/mole) Conc. (mg/L)Conc. (mM) Vitamin B-12 1,355.0 10.72 7.91E−03 Biotin 244.0 0.1566.39E−04 Choline Chloride 140.0 140 1.00E+00 I-Inositol 180.0 1971.09E+00 Niacinamide 122.0 31.5 2.58E−01 Calcium Pantothenate 477.0103.1 2.16E−01 Pyridoxine Hydrochloride 206.0 0.484 2.35E−03 ThiamineHydrochloride 337.0 99.8 2.96E−01 Putrescine 2HCl 161.1 6.66 4.13E−02DL-Lipoic thioctic acid 206.0 4.84 2.35E−02 Sodium Pyruvate 110.0 1,7161.56E+01 Ethanolamine HCl 97.54 76.1 7.80E−01 β-Mercaptoethanol 78.1360.9 7.80E−01 Linoleic Acid 280.48 0.655 2.34E−03 Pluronic F-68 8,350.0780 9.34E−02 Potassium Chloride 74.55 432 5.79E+00 Riboflavin 376.0 3.429.09E−03 Magnesium Chloride 95.21 446 4.69E+00 Anhyd. Magnesium SulfateAnhyd. 120.4 762 6.33E+00 Sodium Selenite 172.9 0.140 8.12E−04 CupricSulfate•5H₂O 249.7 0.1069 4.28E−04 Ferrous Sulfate•7H₂O 278.0 6.512.34E−02 Potassium Nitrate 101.1 0.593 5.86E−03 Zinc Sulfate•7H₂O 287.515.0 5.23E−02 Manganese Sulfate, 169.01 0.00264 1.56E−05 MonohydrateNickelous Chloride, 237.7 0.00186 7.81E−06 6-Hydrate Stannous Chloride2H₂O 225.63 0.001130 5.01E−06 Ammonium Molybdate 1,235.86 0.001931.57E−06 4H₂O Ammonium meta-Vanadate 116.98 0.00913 7.80E−05 Sodiummeta-Silicate 284.2 2.22 7.79E−03 9H₂O EDTA, Iron(III), 367.05 31.28.50E−02 Sodium Salt

The timing and amount of addition of PFFM-3 to the culture occurs asshown in Table 7 below:

TABLE 7 Exemplary DAC HYP Bioreactor Feed Schedule Day PFFM-3 Amount (%of initial mass) 0 0 1 0 2 4-4.14 3 7.8-8.08  4 7.8-8.08  5 7.8-8.08  611-11.38 7 13-13.46 8 15-15.52 9 15-15.52 10 0

Culture pH is maintained at approximately pH 7.0, preferably between pH7.0 and pH 7.1, by automatic control of CO₂ gas and 1 M sodium carbonate(Na₂CO₃) addition. Dissolved oxygen content is allowed to drop toapproximately 30% of air saturation. An oxygen/air mixture is spargedinto the culture to achieve a constant total gas flow rate and dissolvedoxygen is controlled by adjusting the ratio of air to oxygen gases asneeded and by increasing agitation speed after reaching a maximum oxygento air ratio. In another example, agitation is adjusted to maintain aconstant power/volume ratio. A simethicone-based antifoam emulsion isadded to the bioreactor on an as needed basis based on foam level.Samples are taken periodically to test for cell density, cell viability,product concentration, glucose and lactate concentrations, dissolved O₂,dissolved CO₂, pH, and osmolality. The bioreactor culture is harvestedapproximately 10 days post-inoculation. Prior to harvest, the bioreactorcontents are sampled as unprocessed bulk.

7.3.3. Harvest and Cell Removal

Just prior to harvest, the production bioreactor is first chilled to<15° C., then adjusted to a pH of 5.0±0.1 using 0.5 M or 1 M or 2 Mcitric acid, and held for a period of approximately 30-90 or 45-60minutes to flocculate the cells and cell debris prior to transfer to theharvest vessel. The pH-adjusted harvest is then clarified by continuouscentrifugation operated under predefined parameters for bowl speed andflow rate as defined in batch record documentation.

The centrate is filtered through a depth filter followed by a 0.22 μmmembrane filter and collected in a pre-sterilized tank. The cell-freeharvest is adjusted to an approximate pH of 6.4 using a 1-2 M Trissolution and stored at 2-8° C. for further processing. In someinstances, this pH adjustment occurs within 12 hours of the originalbioreactor pH adjustment to pH 5.0.

7.4. DAC HYP Purification

7.4.1. Overview

The DAC HYP purification and formulation process was designed to improveefficiency relative to the ZENAPAX production process and to ensureconsistent clearance of product- and process-related impurities. Thefollowing subsections describe the purification process. Thepurification is based on three chromatography techniques (Protein Aaffinity chromatography, Q Sepharose anion exchange chromatography, andCM-650(M) cation exchange chromatography) in combination with low pHviral inactivation, viral filtration, ultrafiltration/diafiltration, andformulation steps. All of the steps take place in enclosed equipment. Anoutline of the purification process for DAC HYP is presented in FIG. 5and described below.

7.4.2. Protein A Chromatography

The Protein A affinity chromatography step is the first purificationstep in the sequence of downstream operations. This step occurs in oneor more cycles depending on the size of the column, typically two orthree cycles for the column described in Table 8A (i.e., the cell-freeharvest is portioned into two aliquots, and then each aliquot is loadedand eluted separately on the Protein A column). Recombinant Protein Aaffinity chromatography resin specifically binds IgG, separatingantibody from other components of the cell culture harvest.

Following equilibration of the Protein A column with an equilibrationbuffer, the neutralized, cell-free harvest is passed through the columnin order to bind the antibody to the column resin. The equilibrationbuffer is 20 mM sodium citrate, 150 mM sodium chloride, pH 7.0. Thecolumn is loaded to a capacity of no greater than 35 grams antibody(protein) per liter of the packed resin. Following loading, the columnis washed with the equilibration buffer to remove the unbound andloosely bound impurities from the resin, as well as a pre-elution washwith a citrate buffer to adjust the citrate and sodium chlorideconcentration of the column. The citrate buffer is 10 mM sodium citratepH 7.0. The bound antibody is then eluted from the column with a stepchange in pH using an elution buffer of 10 mM sodium citrate at pH 3.5.A summary of the Protein A chromatography conditions is set forth inTable 8A:

TABLE 8A Exemplary DAC HYP Protein A chromatography parameters PARAMETERSET POINT Resin MabSelect Column bed height, cm 10-25, typically 14Column diameter, cm 1-120, depending on scale Operation temperature 5°C. Equilibration/wash buffer 20 mM NaCitrate, 150 mM NaCl, pH 7.0Equilibration volume, CV 5-7 Equilibration flow rate, cm/hr 150-300 Loadflow rate, cm/hr 150-300 Load capacity, grams IgG/L resin <35 Wash flowrate, cm/hr 150-300 Wash volume, CV 7 Pre-Elution conditioning Follow 7CV wash with 2 CV of 10 mM NaCitrate, pH 7.0 and flow rate of 150-300cm/hr Elution buffer 10 mM NaCitrate, pH 3.5 Elution flow rate, cm/hr150-300 Collection criteria 250 mAU-250 mAU (UV detector path-length:5.0 mm) Elution buffer volume post 2 elution, CV Flow direction,equilibration, Down sanitization and storage Equilibration 20 mMNaCitrate, 150 mM NaCl, pH 7.0 Equilibration volume, CV 2-3Equilibration flow rate, cm/hr 150-300 Sanitization buffer 200 mMNaCitrate, 20 mM NaOH, 1% Benzyl Alcohol Sanitization flow rate, cm/hr150 cm/hr for 1.8 CV, then hold for 30 mins. Equilibration for nextcycle 5-7 CV Storage buffer 200 mM sodium citrate, 1% benzyl alcohol, pH7.0 Storage flow rate, cm/hr 150-300 Storage buffer volume, CV 4 CVColumn Storage temp, ° C. 5° C.

As the product elutes off the column, the absorbance of the effluent ata wavelength of 280 nm is monitored and used to guide the collection ofthe product fraction (see FIG. 6).

The use of a sanitization buffer containing sodium hydroxide and benzylalcohol advantageously kills a wide range of microbial organisms whileminimally affecting the quality of the protein A resin. To illustratethis, various sanitization solutions were spiked with variousmicroorganisms and incubated over a period of time. At differentintervals of incubation time, portions of the spiked sanitizationsolutions were neutralized and the microorganism titers were measuredand compared to control. The microbicidal activities are expressed inthe log reduction of the microorganisms over a period of time. Table 8Bshows the reduction of microorganism titers as function of contact timewith sanitization buffer 20 mM sodium hydroxide, 200 mM sodium citrateand 1% benzyl alcohol:

TABLE 8B Reduction of microorganism titers as function of contact timewith sanitization buffer 20 mM sodium hydroxide, 200 mM sodium citrateand 1% benzyl alcohol LRV@ LRV@ LRV@ LRV@ LRV@ Organism 0 min 15 min 30min 60 min 120 min E. coli >5.7 >5.7 >5.7 >5.7 >5.7 (Gram negative) S.aureus 1.1 >5.1 >5.1 >5.1 >5.1 (Gram positive) B. subtilis 2.8 2.7 3.23.1 3.6 (spore forming) (Gram negative) P.aeruginosa >5.0 >5.0 >5.0 >5.0 >5.0 (Gram negative) C. albicans (yeast)4.2 >5.5 >5.5 >5.5 >5.5 A. niger (fungus) −0.2 0.4 0.5 0.8 1.4

Table 8 C shows the reduction of microorganisms by differentsanitization solutions:

TABLE 8C Log₁₀ Reduction of microorganisms by different solutions A B CD E. coli >3.6 >5.7 >4.0 S. aureus >3.6 6.0 0.5 >4.0 Micrococcus lylae3.3 Bacillus sp. (spore forming) B. subtilis −0.3 3.1 0.2 0.12Paenibacillus glucanolyticus −0.01 −0.03 B. cereus 5.0 Pseudomonas sp.P. aeruginosa >3.6 >4.6 Stenotrophomonas altophilia 6.0 Candida albicans(Yeast) 3.1 >5.5 0 >4.8 Aspergillus niger 0.01 0.8 0 >4.7 A = 50 mMNaOH, 0.5M NaCl (60 min) B = 20 mM NaOH, 200 mM sodium citrate, 1%benzyl alcohol (60 min) C = 200 mM sodium citrate, 0.5% benzyl alcohol(48 hrs) D = 2% benzyl alcohol (24 hrs)

The forgoing data shows that sanitization solutions containing benzylalcohol and sodium hydroxide are very effective in killing a widevariety of microorganisms, including gram negative and gram positivebacteria, spore forming bacteria, yeast and fungus. After 30 minutes oftypical sanitization, more than 5 log₁₀ reductions were observed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candidaalbicans. Although the killing of fungus (A. niger) took longer, it israre to have fungus infection in the cell culture fluids. The mostcommon microorganisms isolated in biotech facility are Bacillus,Pseudomonas and Staphylococcus. These are effectively killed by thesanitization solution after 30 minutes of contact time. In comparison,sodium hydroxide or benzyl alcohol alone are not effective in killingall the microorganisms. Moreover, the sodium hydroxide sanitizationsolution does not kill spore forming Bacillus.

7.4.3. Low pH Hold for Viral Inactivation

This step is designed to inactivate low pH-sensitive endogenousvirus-like particles and viruses. The Protein A eluate from each ProteinA cycle is eluted into a collection tank, where 0.5 M HCl is added untila pH 3.5±0.1 is reached. The product is transferred to a hold tank wherethe pH is verified by another pH meter. The low pH hold step is tightlycontrolled at pH 3.5±0.1 or ±0.2 (e.g., pH 3.35-3.64) for 30-120minutes, or 30-240 minutes. After 30±120 minutes hold, the viralinactivated eluate is neutralized to a pH of 7.8±0.1 or ±0.3 (e.g., pH8.05-8.34) using 1 M Tris base, and then transferred through a 0.22 μmfilter into a product pool tank. A summary of the low pH viralinactivation conditions is set forth in the Table 9:

TABLE 9 DAC HYP low pH viral inactivation parameters PARAMETER SET POINTInactivation pH 3.5 Dilution of eluate Dilute to <13 mg/mL and abovetank sample point Dilution buffer 17 mM NaCl or elution buffer pHadjustment buffer 0.5M HCl Inactivation time 120 min at 5° C. or 30 minat ambient temp. Neutralization Buffer 1M Tris Post Neutralization pHTarget 7.8 measured at 25° C.* *Alternatively, the post-neutralizationpH target can be 8.2 at 25° C.

7.4.4. Q Sepharose Anion Exchange Flow Through Chromatography

The Q Sepharose anion exchange chromatography step is used to reduceproduct- and process-related impurities (e.g., nucleic acids, host cellproteins, product aggregates, leached Protein A ligand, etc.) and toprovide additional viral clearance capacity to the purification process.The conductivity and pH of the load are chosen in a manner such that theantibody flows through the column and negatively-charged impurities,such as host cell proteins and cellular DNA, bind to thepositively-charged resin.

The anion exchange column is equilibrated with an equilibration bufferof 20 mM Tris, 20 mM sodium chloride, pH 7.8. The pH-adjusted productfrom the low pH hold step is loaded onto the column to a capacity of nogreater than 60 grams of antibody (protein), or no greater than 30-60grams of antibody (protein), per liter of packed resin. Following thecompletion of loading, unbound antibody and impurities are removed fromthe column with the equilibration buffer.

Collection of the product is guided by monitoring the absorbance of theeffluent at a wavelength of 280 nm (see FIG. 7).

The sanitization flow rate is 100 cm/hr and the hold time is 60 min.

A summary of the Q-sepharose chromatography conditions is set forth inTable 10:

TABLE 10 DAC HYP Q-Sepharose chromatography parameters PARAMETER SETPOINT Resin Q Sepharose FF Column bed height (cm) 10-30, typically 19Column diameter (cm) 1-120, depending on scale Operation temperature 5°C.-25° C. Flow direction for equilibration/load/ Downwash/regeneration/sanitization Equilibration/wash buffer 20 mM Tris, 20mM NaCl, pH 7.8 Equilibration volume (CV)  8 Flow rate (cm/hr) 100 Loadcapacity (g/L) <60 Wash flow rate (cm/hr) 100 Collection criteria 0.25AU-0.25 AU (UV detector path-length: 5.0 mm) Regeneration/sanitizationbuffer 0.5M NaOH, 1M NaCl Regeneration/sanitization volume  1.8 (CV)Regeneration/sanitization hold buffer 0.5M NaOH, 1M NaClRegeneration/sanitization time (min)  60 Storage buffer 12 mM NaOHStorage buffer volume (CV)  4 Column storage temp. (° C.) 5° C.

For some uses, the storage buffer volume set point is 3 and the columnstorage temperature is set at 5°-25° C.

7.4.5. CM-650(M) Cation Exchange Chromatography

This chromatography step is the last step used in the process to reducetrace levels of process- and product-related impurities. In addition toreducing aggregates and cleavage fragments of the antibody, this stepalso reduces process-related impurities such as host cell nucleic acidsand proteins, and leached Protein A.

The column is equilibrated with an equilibration buffer of 20 mM sodiumcitrate, pH 4.5. The anion exchange eluate pool is adjusted to a pH of4.5±0.1 or 0.2 (e.g., 4.35-4.64) using 0.5 M citric acid and loaded ontothe column to a target loading capacity of no greater than 25 or 30grams of antibody (protein) per liter of packed resin. Following thebinding step, the column is washed with the equilibration buffer toremove any unbound, or loosely-bound, impurities from the resin. Thebound antibody is then eluted from the column in a step elution modewith an elution buffer of 20 mM sodium citrate, 75 mM sodium sulfate, pH4.5. Peak collection is guided by monitoring the absorbance of theeffluent at a wavelength of 280 nm (see FIG. 8).

A summary of the CM-650(M) chromatography conditions is set forth inTable 11:

TABLE 11 DAC HYP CM-650(M) chromatography parameters PARAMETER SET POINTResin CM650 (M) Column bed height (cm) 10-30, typically 18 Columndiameter (cm) 1-140, depending on scale Operation temperature 20° C.Equilibration/wash buffer 20 mM NaCitrate, pH 4.5 Equilibration volume(CV)  5 Flow rate (cm/hr) 100 Load preparation Adjust pH of load to 4.5with 0.5M citric acid Load capacity (g/L) ≦30 Wash volume (CV) 3 CV withequilibration buffer Elution buffer 20 mM NaCitrate, 75 mM Na₂SO₄, pH4.5 Cation exchange product pool 1.25 AU up-1.25 AU collection criteria(Based on UV detector path-length of 5.0 mm) IgG concentration dilutedto  8.5 Dilution Buffer* 20 mM NaCitrate, pH 4.5Regeneration/sanitization buffer 0.5M NaOH Regeneration/sanitizationvolume (CV)  1.8 Regeneration/sanitization hold time (min)  60 Storagebuffer 12 mM NaOH Storage buffer volume (CV)  4 *Optionally, dilutionbuffer is not used.

7.4.6. Nanofiltration

The purpose of the nanofiltration step is to provide additional viralclearance capacity to the purification process. The removal of virusesand virus-like particles at this step occurs through a size-exclusionmechanism. The pores of the filter are designed such that the antibodypasses through the filter whereas the virus-like particles and virusesare retained on the upstream side of the filter.

The cation exchange eluate that has been filtered through a 0.22 μm or0.1 μm filter is passed through a small virus-retaining nanofilter,followed by a filter flush with DAC HYP formulation buffer withoutpolysorbate 80 (40 mM succinate, 100 mM sodium chloride, pH 6.0). Thebuffer flush step is applied to recover antibody that remains in theline and filter housing.

A summary of the nanofiltration parameters is set forth in Table 12:

TABLE 12 Nanofiltration parameters PARAMETER SET POINT Pre-filterUltipor Nylon filters Virus Filter V-Pro Magnus 2.2 or NFP Pressure,psig 20-30, typically 25 WFI Flush 100 L/m²* Equilibration buffer and 40mM sodium succinate, wash buffer post load 100 mM NaCl, pH 6.0Equilibration (L) 33 L/m²** Wash buffer volume (L) 33 L/m²† Processcapacity Up to 371 L/m² *or >50 L/m² **or >50 L/m² †or approximately 50L/m²

7.4.7. Ultrafiltration/Diafiltration (UF/DF)

This process step is designed to concentrate the product and exchangethe buffer in the product to the DAC HYP formulation buffer withoutpolysorbate 80. It is operated in a tangential flow mode using a 30 kDanominal molecular weight cutoff membrane. Twoultrafiltration/diafiltration stages are used to produce 150 mg/mLformulation due to the expected product volume at the finalconcentration and relative hold-up volume of each UF system.

The first stage is processed using a large UF system (see FIG. 9). Thenanofiltration filtrate is first concentrated to approximately 30 mg/mLand then diafiltered into exchange buffer (formulation buffer withoutpolysorbate 80). The diafiltered antibody solution is furtherconcentrated to approximately 100 mg/mL, then recovered at aconcentration of approximately 55 mg/mL from the UF/DF system andtransferred through a 0.22 μm filter. The diafiltered antibody solutioncan also be recovered at a concentration of approximately 20-60 mg/mL. Asummary of the parameters of the first stage is set forth in Table 13:

TABLE 13 UF/DF stage 1 parameters UF/DF Membrane: Millipore (Pellicon 2or Pellicon 3, 30 KD MWCO) PARAMETER SET POINT Membrane Loading capacity(g/m2) ≦300   Inlet pressure (psig) 25 Outlet pressure (psig) 15Permeate pressure (psig) Unrestricted/restrictedEquilibration/Diafiltration buffer 40 mM sodium succinate, 100 mM NaClpH 6.0 Concentration during diafiltration (g/L) 30 Diafiltration volume(exchange volumes) 10 Final concentration before product recovery (g/L)~100  Membrane Reuse Yes Filter Flush 40 mM succinate, 100 mM NaCl pH6.0 Sanitization Buffer (pre-use) 0.5M NaOH* Sanitization Time (mins)(pre-use)  60** Sanitization Temperature (° C.) (pre-use) RoomTemperature Sanitization Flush WFI Sanitization Solution (post-use) 0.1MNaOH Sanitization Time (mins) (post-use) 60 Sanitization Temperature (°C.) (post-use) Room Temperature Storage Buffer 0.1M NaOH ProductConcentration (g/L)  70† Product Storage (° C.) 2-20, typically 20 *or0.1M NaOH **or 2 times 40 minutes †or ranging from 20-70

The second stage is processed using a smaller UF system, but with thesame 30 kDa cutoff membrane. The DAC HYP (typically 55 mg/mL) solutionis concentrated to approximately 180 mg/mL, recovered from the UFsystem, and then transferred through a 0.22 μm filter. The UF system isrinsed with formulation buffer without polysorbate 80 and transferredthrough the 0.22 μm filter obtaining the purified drug substance atapproximately 170 mg/mL or approximately 150-170 mg/mL. A summary of theparameters of the second stage is set forth in Table 14:

TABLE 14 DAC HYP UF/DF stage 2 parameters UF/DF Membrane: Millipore(Pellicon 2 or Pellicon 3, 30 KD MWCO) PARAMETER SET POINT MembraneSurface Area (m²) 0.005 to 20.5, depending on scale Inlet pressure(psig)  20 Outlet pressure (psig) Unrestricted Permeate pressure (psig)Unrestricted Equilibration buffer 40 mM sodium succinate, 100 mM NaCl pH6.0 Final concentration before 180 product recovery (g/L)Recirculation/Flush system volume (L) 2 mL to 5 L, depending on scaleRecirculation/flush system buffer 40 mM sodium succinate, 100 mM NaCl,pH 6.0 Sanitization Flush WFI Sanitization Solution (post-use) 0.1M NaOHSanitization Time (mins) (post-use)  60* Storage Buffer 0.1M NaOH 0.2 μmmultimedia filter for 1 × 30 inch filters post final concentration **or2 times 40 minutes

7.5. DAC HYP Formulation

The final process step is the dilution of the purified drug substance toa final target concentration of 150 or 100 mg/L+10%, i.e., a finaltarget concentration of 150±15 mg/mL (in the case of the 150 mg/mLformulation) or 100±10 mg/mL (in the case of the 100 mg/mL formulation)in buffer containing an appropriate concentration of polysorbate 80. Theformulation is performed in stages.

For example, first, the formulation buffer without polysorbate 80 (40 mMsodium succinate, 100 mM sodium chloride, pH 6.0) is added to thepurified drug substance to reach the 90% target volume of formulateddrug substance. Then, a calculated amount of polysorbate 80 dilutionbuffer (40 mM succinate, 100 mM sodium chloride, 1% polysorbate 80, pH6.0) is added to reach the target concentration of 0.03% (w/v)polysorbate 80 in the final formulation. Finally, the product volume isadjusted, using the formulation buffer (made of succinate and succinicacid for the 150 mg/mL formulation, and succinate and HCl for the 100mg/mL formulation) without polysorbate 80, to achieve a final antibodyconcentration of 150±15 mg/mL (preferably 150±8 mg/mL). The 100 mg/mLdrug product is formulated in a similar manner to a final concentration100±10 mg/mL (preferably 100±5 mg/mL).

The formulated drug substance is filtered through a 0.22 μm filter intoa BioProcess Container™ (BPC®) bag (or equivalent) which is placedinside a semi-rigid cylindrical support. The support encloses the BPCwith a lid and provides a protective barrier between the flexible bagand the external environment. The formulated drug substance is stored at2-8° C. in an access-controlled cooler for drug product fill/finishoperations.

A summary of the formulation conditions is set forth in Table 15:

TABLE 15 150 mg/ml DAC HYP formulation conditions 0.2 μm filter for postformulation pool PARAMETER SET POINT Dilution buffer 40 mM sodiumsuccinate, 100 mM NaCl, pH 6.0 Polysorbate 80 Stock Solution 40 mMsodium succinate, 100 mM NaCl, pH 6.0, 1% (w/v) polysorbate 80 FinalPolysorbate 80 concentration (w/v) % 0.03 Final Daclizumab concentration(mg/mL) 100 or 150 Product Storage (° C.) 2-8, commonly 5

1 mL of the drug product is filled into vials or a syringe. A summary ofthe components of the finished 150 mg/mL and 100 mg/mL products have thecomponents shown in Table 16 (all quantities are nominal values):

TABLE 16 Composition of the 150 mg/mL and 100 mg/mL DAC HYP Drug ProductFormulations Drug product: Drug product: Ingredient 150 mg/mL 100 mg/mLDAC HYP 150 mg 100 mg Sodium Succinate  5.9 mg  6.5 mg Succinic Acid 0.4 mg Not applicable Sodium Chloride  5.8 mg  5.8 mg Polysorbate 80 0.3 mg  0.3 mg Hydrochloric Acid Not applicable As needed foradjustment to pH 6.0 Sodium Hydroxide As needed for adjustment As neededfor adjustment to pH 6.0 to pH 6.0 Water for Injection As needed forfinal As needed for final volume volume

7.6. Characterization of DAC HYP Drug Substance

DAC HYP is glycosylated at amino acid 296 of both heavy chain subunits,with the major oligosaccharide form existing as a core fucosylatedbiantennary structure lacking terminal galactose.

The N-terminus of DAC HYP heavy chain exists as three major forms: 1)N-terminal glutamine (predicted from the DNA sequence), 2) N-terminalpyroglutamate (from the cyclization of N-terminal glutamine), and 3)N-terminal valine, histidine and serine residues in addition to thepredicted N-terminal glutamine residue (results from incomplete cleavageof the signal peptide).

The C-terminus of DAC HYP heavy chain exists with and without theC-terminal lysine residue. The major form lacks the C-terminal lysineresidue, resulting in a C-terminal glycine.

DAC HYP has a calculated molecular weight of 144 kDa based on theprimary amino acid composition defined by the nucleotide sequence. Thecorresponding molecular weight of the reduced heavy chain is 48.9 kDaand the reduced light chain is 23.2 kDa; these weights do not accountfor carbohydrate content or post-translational modifications.

DAC HYP binding is highly specific for CD25, which is expressed onactivated but not on resting T and B lymphocytes. DAC HYP binding toCD25 on these activated cells blocks the binding of IL-2 to CD25 andsubsequent formation of the high affinity IL-2 receptor complex.Consequently, IL-2-induced proliferation of the activated cells isblocked. The observed and potential therapeutic efficacy of DAC HYP isbelieved to rest in large part on its inhibitory effect on theproliferation of activated autoreactive T-cells. However, DAC HYP mightalso exert a therapeutic effect through its blocking effect on otherCD25-bearing cell types such as eosinophils.

To confirm that high concentration 150 mg/ml DAC HYP formulations weresuitable for clinical investigations, a comprehensive physicochemicaland biological evaluation was performed to characterize and compare twobatches of DAC HYP 150 mg/mL drug substance, referred to herein as Batch1 and Batch 2 (or Batch 150-1 or Batch 150-2, respectively), toReference Standard lot RS0801, which is from a lot of DAC HYP 100 mg/mLmanufactured at the 10,000 L scale.

The results demonstrate that the DAC HYP drug product 150 mg/mL lots areof high purity, are comparable to the 100 mg/mL lots, and are suitablefor use in clinical studies. A summary of these characteristics is shownin Table 17:

TABLE 17 Purity characteristics of DAC HYP (all concentrations arenominal) 150 mg/mL lots 100 mg/mL lots Useful Criteria Category Test (2batches) (24 batches) (for 150 mg/ml) Quality Color, appearanceColorless, clear to Colorless, clear to Colorless, clear to and clarityslightly opalescent slightly opalescent slightly opalescent liquid,essentially free liquid; no visible liquid, essentially free of visibleparticles particles of visible particles pH determination 6.0 at 25° C.6.0-6.1 at 25° C. 5.8-6.2 at 25° C. Product 143-148 mg/mL 96.0-103 mg/mL135-165 mg/mL concentration by UV spectroscopy Cation ExchangeChromatogram Not available Chromatogram Chromatography consistent withconsistent with reference reference Identity Anti-idiotype Identifies asIdentifies as Identifies as ELISA daclizumab daclizumab daclizumabCation exchange Not applicable Chromatogram Not applicablechromatography consistent with reference Purity Size exclusion 99.3% to99.4% main 99.2% to 99.6% main ≧95% main peak chromatography peak peak≦3% aggregate 0.6% to 0.7% 0.4% to 0.8% aggregate aggregate SDS-PAGE97.3% purity 95.8%-97.0% purity ≧93% purity (colloidal blue stain)reduced SDS-PAGE (silver Sample staining Sample staining Sample stainingstain) reduced and consistent with consistent with consistent withnon-reduced reference reference reference DNA content <0.1 pg/mg<0.01-<0.06 pg/mg ≦0.25 pg/mg Residual protein A <1 ppm-1 ppm <1 ppm ≦30ppm (based on weight of protein A relative to weight of antibody) Hostcell protein <1 ppm (weight <1 ppm-5 ppm ≦50 ppm basis) (weight basis)(based on weight of host cell protein relative to weight of antibody)Potency Binding potency 86%-105% relative 78%-129% relative 70%-130%relative potency potency potency Functional 95%-101% relative 73%-121%relative 70%-130% relative potency potency potency potency SafetyBacterial <0.01 EU/mg <0.01 EU/mg ≦0.751 EU/mg endotoxins Bioburden 0CFU/100 mL 0 CFU/10 mL ≦10 CFU/100 mL 0 CFU/100 mL ExcipientsPolysorbate 80 0.026%-0.029% w/v 0.027%-0.034% w/v 0.024%-0.036% w/vOsmolality 281-290 mOsm/Kg 288-299 mOsm/Kg 267-327 mOsm/Kg

7.6.1. Color, Appearance, and Clarity

The appearance of DAC HYP drug substance is assessed by visuallyexamining the color and clarity of the solution in direct light againsta black background and white background without magnification. Thesolution is also evaluated for the presence of visible particles. Thetypical appearance of various lots of DAC HYP drug product is describedin Table 17.

7.6.2. pH Determination

The pH of DAC HYP is determined in accordance with the U.S. PharmacopeiaProtocol No. (791). The pH ranges of various lots of DAC HYP drugproduct are summarized in Table 17.

7.6.3. Product Concentration by UV Spectroscopy

The concentration of DAC HYP is determined by UV spectroscopy. DAC HYPsamples are diluted gravimetrically with buffer. The UV absorbance ofeach diluted sample solution is measured at 278 nm against a bufferblank. The protein concentration of the sample is calculated using theabsorptivity coefficient for DAC HYP. The protein concentrations ofvarious lots of DAC HYP drug product are summarized in Table 17.

7.6.4. N-Terminal Sequencing

DAC HYP 150 mg/mL lots were evaluated by N-terminal sequencing. Thesamples were analyzed using an automated Edman degradation sequencinginstrument.

The expected amino acid sequence of the light chain through the first 15residues, DIQMTQSPSTLSASV (SEQ ID NO:13), was confirmed for all samples.

The majority of heavy chain in DAC HYP is blocked by a pyroglutamate(pE) residue that will not produce an N-terminal heavy chain sequence.The next most prevalent N-terminal heavy chain sequence in DAC HYPbegins with a valine, histidine, serine (VHS) sequence, resulting fromthe lack of processing the three terminal residues of the heavy chainsignal peptide. Fourteen of the first fifteen N-terminal residues wereconfirmed for the VHS heavy chain sequence (VHSQVQLVQSGAEVK (SEQ ID NO:14)) in all samples. The fourth residue, glutamine, could not beconfirmed due to the large amount of glutamine detected from LC in thepreceding sequencing cycle. Evidence of heavy chain with N-terminalglutamine was also present in all samples. This sequence is a result ofthe native N-terminal heavy chain glutamine residue not undergoingcyclization to the pyroglutamate form. The N-terminal sequencing resultsfor the 150 mg/mL lots are consistent with the sequences predicted fromthe heavy and light chain coding sequences. Comparable results wereobtained for the 100 mg/mL lots.

7.6.5. Heavy and Light Chain Mass Analysis

The molecular masses of the heavy chain and light chain of the DAC HYP150 mg/mL lots and Reference Standard RS0801 were evaluated by liquidchromatography mass spectrometry (LC-MS) analysis. All lots weredeglycosylated with the amidase PNGaseF, reduced with dithiothreitol,alkylated with iodoacetic acid, and separated by reversed phasechromatography. Theoretical heavy and light chain masses were calculatedfrom the protein sequence. The observed masses of the samples werewithin 1 Da of the calculate masses, as shown in Table 18 below:

TABLE 18 Heavy and Light Chain Mass Results LC (Da) HC (pE, -K)(Da) HC(VHS, -K) (Da) Theoretical Mass 23,505 49,356 49,697 RS0801 23,50549,357 49,696 Batch 150-1 23,505 49,357 49,696 Batch 150-2 23,504 49,35749,696

As described in the preceding subsection, the two most prevalent formsof DAC HYP heavy chain are known to contain an N-terminal pyroglutamate(pE) residue or a valine, histidine, serine (VHS) sequence and lackC-terminal lysine. The molecular weights obtained for the twopredominant heavy chain variants and the light chain in the 150 mg/mLlots were comparable to those of Reference Standard RS0801 andconsistent with the masses predicted from the protein sequences.

Together with the peptide mapping results presented in the followingsubsection, the heavy and light chain mass results confirm the presenceof the expected light chain and heavy chain primary structures in theDAC HYP 150 mg/mL lots.

7.6.6. Peptide Mapping

DAC HYP 150 mg/mL lots and Reference Standard RS0801 (DAC HYP producedfrom a 100 mg/mL drug substance lot manufactured at the 10,000 L scale)were evaluated using reversed phase HPLC peptide mapping. All lots werereduced with dithiothreitol, alkylated with iodoacetic acid, andenzymatically digested with trypsin. The resulting peptides wereseparated by reversed phase chromatography and detected by ultravioletabsorbance at 215 nm to generate peptide maps.

To verify the primary amino acid sequence, the peptide maps of the 150mg/mL lots were compared to that of Reference Standard RS0801. Peptidescorresponding to ninety eight percent of the expected heavy and lightchain residues have previously been identified by mass spectrometry inthe peptide map of Reference Standard RS0801. The residues that have notbeen accounted for in the peptide map are single amino acids or residein very polar dipeptides, and are not expected to be retained by thereversed phase column. Masses consistent with pyroglutamate, glutamine,and the VHS sequence at the N-terminus of the heavy chain N-terminalpeptide have been identified in the reference standard. DAC HYP containsa consensus site for N-linked glycosylation in the Fc portion of theheavy chain at Asn296 and masses consistent with linked complex corebiantennary oligosaccharide structures have been identified for thepeptide containing the Asn296 residue.

Peptide maps comparing the DAC HYP 150 mg/mL lots to Reference StandardRS0801 are shown in FIG. 10 (0 to 60 minutes), FIG. 11 (55 to 115minutes), and FIG. 12 (110 to 170 minutes). The peptide maps of the DACHYP 150 mg/mL lots are comparable to those of the reference standard andconfirm the presence of the expected primary structure in the 150 mg/mLlots.

7.6.7. Circular Dichroism Spectroscopy

DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed byfar ultraviolet circular dichroism spectroscopy (far-UV CD) to evaluatesecondary structure. Prior to analysis samples were diluted with waterto a final protein concentration of 0.2 mg/mL. Spectra were acquiredfrom 195 to 260 nm using a 0.1 cm cell and the signal obtained wasconverted to molar ellipticity after buffer subtraction.

The overlaid far-UV CD spectra of DAC HYP 150 mg/mL lots Batch 1 andBatch 2 and Reference Standard RS0801 are shown in FIG. 13. The spectraof all lots show a positive band at approximately 202 nm and a negativeband at approximately 217 nm. The negative band at 217 nm ischaracteristic of a P sheet conformation, the predominant conformationof IgG molecules. The far-UV CD spectra of the lots were similar, whichis supportive of a uniform secondary structure among the DAC HYP 150mg/mL lots and Reference Standard RS0801.

7.6.8. Ultraviolet Spectroscopy

DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed byultraviolet (UV) spectroscopy to evaluate tertiary structure. Prior toanalysis samples were diluted with formulation buffer (40 mM succinate,100 mM sodium chloride, 0.03% polysorbate 80, pH 6.0) to a final proteinconcentration of 0.5 mg/mL. Spectra were acquired from 250 to 350 nmusing a 1 cm path length quartz cuvette and normalized to an absorbanceof 1.0 at 280 nm.

The overlaid zero-order and second derivative UV spectra (calculatedfrom the smoothed zero-order data) are shown in FIGS. 14A and 14B,respectively. The zero-order and second derivative spectra of all lotsevaluated were superimposable, which is supportive of a uniform tertiarystructure among the DAC HYP 150 mg/mL lots Batch 1 and Batch 2 andReference Standard RS0801.

7.6.9. Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed using a porous silicacolumn with an aqueous mobile phase and ultraviolet absorbance detectionat 280 nm. In particular, 15 μL test sample (20 mg/mL antibody inelution buffer) was analyzed at room temperature on a 7.8 mm×30 cm TSKG3000SW_(XL) column (Tosoh Biosciences, part no. 601342) equipped with a0.5 m pre-column filter (Upchurch, part no. A-102X) using an isocraticgradient of elution buffer (200 mM KPO₄, 150 mM KCl, pH 6.9) at a flowrate of 1 mL/min.

As shown in FIG. 15A (full scale) and FIG. 15B (expanded scale), thechromatographic profiles of the DAC HYP 150 mg/mL lots were comparableto that of Reference Standard RS0801. All lots exhibited the same majorpeak corresponding to daclizumab monomer and a minor aggregate peak.Aggregate results for DAC HYP 150 mg/mL lots were comparable to those ofDAC HYP 100 mg/mL lots as shown in Table 19 below:

TABLE 19 Percentage Aggregate Results 100 mg/mL (n = 24 lots) Average0.6 Standard Deviation 0.1 Minimum 0.4 Maximum 0.8 150 mg/mL Batch 150-10.6 Batch 150-2 0.7

The 150 mg/mL lots and Reference Standard RS0801 were analyzed using SECwith multi-angle light scattering detection (SEC-MALS) to determine themolecular weight of the aggregate peak. For all lots, the molecularweight obtained for the aggregate peak was approximately 300 kDa, whichis consistent with antibody dimer.

Aggregate formation in DAC HYP was monitored over an 18-month period.The level of aggregates in the formulation rose, but the percentage ofaggregates plateaued and did not exceed approximately 1.5% when storedat 5° C. for 18 months (see FIG. 16). New data with new batchesindicates that approximately 1.8-1.9% aggregates may appear at 6 weekswhen stored at 5° C., but for all samples tested, less than 3%aggregates formed over a period of 5 years when stored at 5° C.

7.6.10. Sedimentation Velocity Analytical Ultracentrifugation

The monomer and aggregates in DAC HYP 150 mg/mL and 100 mg/mL lots werecharacterized using sedimentation velocity analyticalultracentrifugation (SV-AUC). The sedimentation coefficient value andrelative abundance for the monomer and each of the aggregates arepresented in Tables 20 and 21 below.

TABLE 20 Sedimentation Coefficient Values for Monomer and Aggregates (inSvedbergs) Monomer Dimer Trimer Tetramer 100 mg/mL Average 6.14 8.4810.6 12.1 (n = 8 lots) Range 6.11-6.18 8.36-8.67 10.3-10.8 11.8-12.7 150mg/mL Batch 6.16 8.52 10.4 11.6 150-1 Batch 6.13 8.33 10.3 11.9 150-2

TABLE 21 Relative Abundance of Monomer, Dimer, Trimer, and Tetramer % %% % Monomer Dimer Trimer Tetramer 100 mg/mL Average 97.5 1.8 0.4 0.2 (n= 8 lots) Range 96.7-98.5 1.3-2.2 0.2-0.7 0.1-0.4 150 mg/mL Batch 150-197.6 1.9 0.4 0.1 Batch 150-2 96.9 2.3 0.5 0.3

Monomer was the major component observed in each of the lots. Thesedimentation coefficient of the monomer peak was highly consistentamong the lots indicating that the conformation of the monomer iscomparable between the 150 mg/mL and 100 mg/mL lots. The monomer contentof the 150 mg/mL lots was comparable to that of the 100 mg/mL lots.

The predominant aggregate species in each of the lots had asedimentation coefficient consistent with antibody dimer. This isconsistent with SEC-MALS results, which indicate that the SEC aggregatepeak is composed primarily of antibody dimer (see preceding subsection).Low levels of two larger aggregate species that had sedimentationcoefficients consistent with trimer and tetramer were also observed ineach of the lots by AUC. The dimer, trimer, and tetramer content of the150 mg/mL lots was comparable to that of the 100 mg/mL lots.

7.6.11. Quantitative Reduced SDS-PAGE

Purity was determined by SDS-PAGE using 4-20% (typically 14%)tris-glycine gels with Colloidal blue stain. Samples were analyzed underreducing conditions with a sample load of 10 g. Purity was calculated bydividing the sum of the heavy chain and light chain band area by thetotal band area as measured by densitometry.

As shown in Table 22 below, the 150 mg/mL lots are of high purity andcomparable to the 100 mg/mL lots:

TABLE 22 Quantitative Reduced SDS-PAGE Results % Purity 100 mg/mL (n = 7lots) Average 96.4 Standard Deviation 0.5 Minimum 95.8 Maximum 97.0 150mg/mL Batch 150-1 97.3 Batch 150-2 97.3

7.6.12. Qualitative SDS-PAGE

Purity of DAC HYP was assessed by both reduced and non-reduced gelelectrophoresis. Precast 14% or 8-16% Tris-glycine gels were used forthe analysis. Aliquots from the two batches of 150 mg/mL DAC HYPformulation were compared to a reference batch, as previously described.Reduced and non-reduced gels analyzing the purity of DAC HYP are shownin FIG. 17. The band pattern of the 150 mg/mL lots was comparable tothat of Reference Standard RS0801, with no new bands detected in the 150mg/mL lots.

7.6.13. Cation Exchange Chromatography

The charge isoform distribution of the DAC HYP 150 mg/mL lots and 100mg/mL lots were evaluated using cation exchange chromatography (CEX).CEX was performed using a nonporous, carboxylate functionalized, weakcation exchange column with detection at 220 nm. 100 μL of test sample(1 mg/mL antibody dissolved in Buffer A) was resolved at roomtemperature on a ProPac WCX-10 column (Dionex Coporation) equipped witha ProPac WCX-10G guard column (Dionex Corporation) using the followingseparation gradient (column is equilibrated with Buffer A):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0.0 100 0 1 60.0 4060 1 80.0 0 100 1 85.0 0 100 1 85.1 100 0 1 100.0 100 0 1 Buffer A = 15mM sodium phosphate, pH 5.9 Buffer B = 250 mM NaCl, 15 mM sodiumphosphate, pH 5

As shown in FIG. 18, the CEX chromatograms of the 150 mg/mL lots areconsistent with those of Reference Standard RS0801, with no new chargeisoforms detected in the 150 mg/mL lots. The five major isoforms presentin the CEX chromatograms are due to heterogeneity at the heavy chainN-terminus and include: 1) two pyroglutamate residues (pE/pE); 2) onepyroglutamate residue and one glutamine residue (pE/Q); 3) onepyroglutamate residue and one VHS sequence (truncated VHS signal peptidepreceding the N-terminal glutamine residue of the mature heavy chain)(pE/VHS); 4) one glutamine residue and one VHS sequence (Q/VHS); 5) twoVHS sequences (VHS/VHS). C-terminal lysine (K) isoforms are alsoresolved and identified in FIG. 18. Each of the N-terminal isoformsdescribed above may exist as different C-terminal isoforms (0, 1, or 2K). Because of the complexity of the mixture, the C-terminal lysineisoforms are only clearly resolved and measurable for the major pE/pEand pE/VHS isoforms using the described method.

Quantitative N- and C-terminal isoform results are provided for the 150mg/mL and 100 mg/mL lots in Tables 23 and 24, respectively, where thereported percentage is based upon the percentage of the area under thecurve (AUC) of the specific peak as compared to the total AUC of allpeaks:

TABLE 23 CEX Results - N-Terminal Isoforms % pE/pE % pE/Q % pE/VHS %Q/VHS % VHS/VHS 100 mg/mL Average 38 11 40 7 5 (n = 24 lots) StandardDeviation 2.8 0.8 2.0 1.1 3.8 Minimum 31 9 34 4 1 Maximum 42 12 42 9 17150 mg/mL Batch 150-1 31 9 31 12 17 Batch 150-2 32 9 31 11 17 pE/pE =Two Pyroglutamate Residues; pE/Q = One Pyroglutamate Residue, OneGlutamine Residue; pE/VHS = One Pyroglutamate Residue, One TruncatedSignal Peptide; Q/VHS = One Glutamine Residue, One Truncated SignalPeptide; and VHS/VHS = Two Truncated Signal Peptides.

TABLE 24 CEX Results - C-Terminal Isoforms % 0K % 1K % 2K 100 mg/mLAverage 78 16 6 (n = 24 lots) Standard Deviation 2.5 1.7 1.0 Minimum 6914 5 Maximum 80 22 10 150 mg/mL Batch A 73 19 8 Batch B 74 19 8 0K = NoC-terminal lysine residue on either heavy chain 1K = C-terminal lysineresidue on one heavy chain 2K = C-terminal lysine residue on both heavychains

7.6.14. Oligosaccharide Mapping

The oligosaccharide distributions of the DAC HYP 150 mg/mL and 100 mg/mLlots were evaluated by oligosaccharide mapping. N-linkedoligosaccharides were released enzymatically from heavy chain Asn296using the amidase PNGaseF. The oligosaccharides were subsequentlyderivatized with a fluorescent label (in this case anthranilic acid) andseparated from the antibody via a nylon membrane. The derivatized,cleaved N-linked glycans were resolved at 50° C. on a 250×4.6 mmpolymeric-amine bonded Asahipak Amino NH₂P-504E column (5 μm particlesize, Phenomenex, cat. No. CHO-2628) with fluorescent detection, usingthe following elution gradient (using a sample injection volume of 100μL; column is equilibrated with 85% Buffer A/15% Buffer B):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0 85 15 1 2 85 15 110 80 20 1 60 55 45 1 70 5 95 1 75 5 95 1 76 85 15 1 90 85 15 1 Buffer A= 1% v/v tetrahydrofuran, 2% v/v acetic acid in acetonitrile Buffer B =1% v/v tetrahydrofuran, 5% v/v acetic acid, 3% v/v triethylamine inwater

Chromatograms comparing the 150 mg/mL lots to Reference Standard RS0801are shown in FIG. 19. Oligosaccharide peaks constituting at least 1.0%of the total peak area are labeled and reported below in Table 25:

TABLE 25 Oligosaccharide Results % % % % G0-GlcNAc G0 Peak 3 G1 100mg/mL Average 8.6 86.3 1.5 2.0 600 L (n = 22 lots) Minimum 6.9 84.6 1.41.5 Maximum 10.6 88.2 1.6 2.3 100 mg/mL Batch 100-A 11.2 82.3 1.7 3.2 10kL (n = 2 lots) Batch 100-B 9.0 83.7 1.7 3.7 150 mg/mL Batch 150-1 7.385.6 1.6 3.8 10 kL (n = 2 lots) Batch 150-2 7.2 85.6 1.6 3.7 G0-GlcNAc:Biantennary core structure with fucose attached to the N-linked N-acetylglucosamine, one N-acetyl glucosamine on one branch of the corestructure and no terminal galactose. G0: Biantennary core structure withfucose attached to the N-linked N-acetyl glucosamine, one N-acetylglucosamine on each branch of the core structure and no terminalgalactose. G1: Biantennary core structure with fucose attached to theN-linked N-acetyl glucosamine, one N-acetyl glucosamine on each branchof the core structure and terminal galactose on one branch of the corestructure.

All lots consist primarily of G0 and G0-GlcNAc (G0 lacking GlcNAc on onearm of the biantennary structure), which is representative of the DACHYP process. Sialylated oligosaccharides elute at approximately 68minutes and are below 1.0% in all lots tested. The uncharacterizedoligosaccharide referred to as Peak 3 was present in similar abundancein all lots tested.

7.6.15. Oxidation

DAC HYP lots were evaluated for potential methionine oxidation, bymonitoring oxidized and non-oxidized tryptic peptides present in thepeptide maps. The peak areas of the non-oxidized and oxidized forms ofeach methionine containing peptide were determined using the massspectra extracted ion chromatograms. For each methionine residue, thepercent oxidized methionine was calculated by dividing the mass spectrapeak area of the oxidized peptide by the sum of the peak areas of theoxidized and non-oxidized peptides.

As shown in Table 26 below, methionine oxidation results for the 150mg/mL lots and five 100 mg/mL lots were comparable:

TABLE 26 Oxidation Results (LC = light chain; HC = heavy chain; M =methionine) Percent Oxidation Lots LC M4 LC M32 HC M34 HC M 81 HC M251HC M427 100 mg/mL Batch 100-1 0.8 0.6 0.5 0.6 3.8 1.1 Batch 100-2 0.90.7 0.6 0.7 3.7 1.3 Batch 100-3 0.8 0.7 0.5 0.7 4.0 1.3 Batch 100-4 0.80.6 0.4 0.7 4.8 1.8 Batch 100-5 0.6 0.6 0.3 0.6 4.6 1.5 Average 0.8 0.70.5 0.7 4.2 1.4 Standard Deviation 0.1 0.1 0.1 0.1 0.5 0.3 Minimum 0.60.6 0.3 0.6 3.7 1.1 Maximum 0.9 0.7 0.6 0.7 4.8 1.8 150 mg/mL Batch150-1 0.6 0.6 0.4 0.5 3.5 0.9 Batch 150-2 0.8 0.7 0.4 0.7 4.3 1.6

Heavy chain Met251 and Met427 are the most labile and exhibits thegreatest degree of oxidation. Among the lots tested concurrently toevaluate comparability, oxidation levels for Met251 and Met427 did notexceed 4.8% and 1.8%, respectively.

7.6.16. Binding Potency (CD25 Binding)

DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for binding to thealpha subunit of the IL-2 receptor (CD25) via ELISA as a measure ofpotency as part of release testing. Microtiter plates were immobilizedwith soluble CD25 and incubated with varying amounts of DAC HYP. BoundDAC HYP was detected using a horseradish peroxidase-conjugated goatanti-human IgG antibody in tandem with 3,3′,5,5′-tetra-methyl benzidinesubstrate. Resulting absorbance values were plotted against the log₁₀ ofDAC HYP concentration using a 4-parameter fit and percent relativepotency values were generated using parallel line analysis.

Drug substance results are summarized the table below:

TABLE 27 Potency by ELISA Results Potency (% of Reference Standard) 100mg/mL (n = 24 lots) Average 101 Standard Deviation 14 Minimum 78 Maximum129 150 mg/mL Batch 150-1 86 Batch 150-2 105

The binding potency results of the 150 mg/mL lots were comparable tothose of the 100 mg/mL lots.

7.6.17. Surface Plasmon Resonance (CD25 Binding)

Surface plasmon resonance analysis was performed to determine theaffinity constant (K_(D)) for the binding interaction of DAC HYP to thealpha subunit of the IL-2 receptor (CD25).

Goat anti-human IgG Fc antibody was immobilized on a chip surface tocapture DAC HYP samples, after which soluble CD25 was injected atvarious concentrations in duplicate over captured DAC HYP using anautomated method. Binding data were collected and corrected using areference flow cell and buffer blank, and fit with BIA Evaluationsoftware using a 1:1 Langmuir model to obtain equilibrium constants.

Results for DAC HYP 150 mg/mL lots and Reference Standard RS0801 aresummarized in Table 28:

TABLE 28 Surface Plasmon Resonance Results Lots Ka (1/M*s) Kd (1/s) KD(M) % RS KD RS0801 (“RS”) 3.3 × 10⁵ 1.5 × 10⁻⁴ 4.4 × 10⁻¹⁰ NotApplicable Batch 150-1 3.3 × 10⁵ 1.5 × 10⁻⁴ 4.6 × 10⁻¹⁰ 96 Batch 150-23.4 × 10⁵ 1.5 × 10⁻⁴ 4.4 × 10⁻¹⁰ 100

The association constant (k_(a)), dissociation constant (k_(d)), andaffinity constant (K_(D)) values of the 150 mg/mL lots were comparableto those of Reference Standard RS0801.

7.6.18. Functional Potency

DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for functionalpotency as part of release testing. The functional potency assaymeasures the inhibition of IL-2 induced T-cell proliferation by bindingof DAC HYP to the alpha subunit of the IL-2 receptor (CD25). In thepresence of IL-2, varying amounts of DAC HYP were incubated with KIT-225K6 cells (Hori et al., 1987, Blood 70:1069-1072) expressing the IL-2receptor. Inhibition of T-cell proliferation by DAC HYP was subsequentlydetected using alamar blue. Resulting fluorescence values were plottedagainst the log₁₀ of DAC HYP concentrations using a 4-parameter fit andpercent relative potency values were generated using parallel lineanalysis.

The functional potency results are summarized in Table 29:

TABLE 29 Functional Potency Results Potency (% of Reference Standard)100 mg/mL (n = 24 lots) Average 98 Standard Deviation 12 Minimum 73Maximum 121 150 mg/mL Batch 150-1 101 Batch 150-2 95

The functional potency results of the 150 mg/mL lots were comparable tothose of the 100 mg/mL lots.

7.6.19. Antibody Dependent Cellular Cytotoxicity

Two lots of DAC HYP 150 mg/mL formulations were evaluated relative tothat of Reference Standard RS0801 100 mg/mL DAC HYP.

IL-2 receptor expressing KIT-225 K6 cells were labeled with ⁵¹Cr, andsubsequently incubated with DAC HYP. Human effector cells (PBMC) wereadded in varying amounts to achieve different effector to target cell(KIT-225 K6) ratios. Fc receptor bearing monocytes interact with the DACHYP Fc domain and subsequently cause target cell lysis. The degree ofcytotoxicity was determined by measuring the release of ⁵¹Cr from targetcells and was expressed as a percentage of maximum cell lysis.

PBMCs from multiple donors were utilized for each sample. For eachdonor, the percent ADCC activity of the sample was calculated relativeto that of Reference Standard RS0801 based on percent cytotoxicity. ADCCresults are summarized in Table 30 below:

TABLE 30 ADCC Results % Cytotoxicity (Relative to Reference StandardRS0801) Standard Lot Donor #1 Donor #2 Donor #3 Average Deviation Batch150-1 93 79 76 83 9 Batch 150-2 93 92 80 88 7

Response curves for the 150 mg/mL lots, Reference Standard RS0801,positive and negative control antibodies and a control without antibody(for Antibody Independent Cellular Cytotoxicity or AICC) are shown inFIG. 20.

The ADCC activity of the 150 mg/mL lots was comparable to that ofReference Standard RS0801.

7.6.20. Residual Protein A

Residual Protein A may be determined by an ELISA method, wherestandards, sample controls, a plate blank, and test samples are dilutedwith a denaturing buffer and placed into a boiling water bath todissociate Protein A and denature and precipitate daclizumab. Afterboiling, standards, controls, and samples are cooled, centrifuged, andadded to a micro-titer plate coated with polyclonal anti-Protein Acapture antibody. Residual Protein A present in the samples is thendetected using a biotinylated anti-Protein A antibody in tandem withstreptavidin alkaline phophatase and P-nitrophenyl phosphate (PNPP)substrate. The plate is analyzed in a spectrophotometric plate readerand a log-log standard curve is generated, against which theconcentration of Protein A is determined. Test sample results arereported in parts per million (ppm) units. Parts per million results arecalculated by dividing the ng/mL Protein A result by the antibodyconcentration of the test sample in mg/mL.

7.6.21. DNA Content

Detection of mouse DNA is determined at a contract laboratory using aquantitative polymerase chain reaction (Q-PCR) test method. In themethod, the sample is subjected to DNA extraction. The sample extract isthen analyzed by Q-PCR using mouse specific primers and probe to amplifya specific fragment of a repetitive element of mouse DNA. Amplificationof the DNA results in a fluorescence signal that is detected. The DNA inthe sample is quantitatively measured by comparison to a standard curvegenerated using known amounts of mouse DNA. Results are expressed inpicograms of DNA per milligram of antibody. The average DNA content invarious lots of DAC HYP drug product are summarized in Table 17.

7.6.22. Host Cell Proteins (HCP)

Residual host cell proteins in the product are quantified using acommercially available kit. An affinity purified goat polyclonalantibody to NS0 cell lysate is used for both the capture and detectionof NS0 HCP. The HCP standard is produced by collecting cell free harvestmaterial from a mock production run. A standard curve is prepared usingan HCP working standard and samples containing HCP are serially dilutedto target the range of the standard curve. Standards, sample controls,and test samples are added to an anti-NS0 HCP polyclonal antibody coatedplate. Host cell proteins are then detected with an anti-NS0 HCPpolyclonal antibody conjugated to horseradish peroxidase (HRP) in tandemwith 3,3′,5,5′-tetra-methyl benzidine (TMB) substrate. The plate is thenanalyzed in a spectrophotometric plate reader and a four parameter curvefit is generated to quantitate the amount of HCP in the samples.

The results for the NS0 HCP ELISA assay are reported in parts permillion (ppm) units. Parts per million results are calculated bydividing the ng/mL HCP result by the antibody concentration in mg/mL.The average HCP of various lots of DAC HYP drug product are summarizedin Table 17.

7.6.23. Polysorbate 80 Concentration

Polysorbate 80 is quantitated using a spectrophotometric method that isbased on the formation of a colored cobaltthiocyanate complex withpolysorbate 80. A standard curve is constructed using a series ofpolysorbate 80 standards. The polysorbate 80 concentration in the sampleis determined from the standard curve. The ranges of polysorbateconcentrations of various lots of DAC HYP drug product are summarized inTable 17.

7.6.24. Osmolality

Osmolality is measured using a vapor pressure depression osmometer.Prior to sample analysis the osmometer is calibrated using osmolalitystandards that bracket the expected osmolality of the sample. Theosmolality ranges of various lots of DAC HYP drug product are summarizedin Table 17.

7.6.25. Conclusions

The physicochemical and biological analyses conducted provide acomprehensive evaluation of DAC HYP 150 mg/mL and DAC HYP 100 mg/mLformulations. The physicochemical and biological characteristics of alllots tested to date are comparable.

For all DAC HYP lots, the first 15 amino acids of the heavy and lightchains determined by N-terminal sequencing, peptide maps and molecularweight analyses were consistent with the daclizumab gene sequences.

The aggregate levels and size distribution of aggregate species in all150 mg/mL and 100 mg/mL lots tested, as determined by SEC-MALS andSV-AUC were comparable, as were their purity as tested by gelelectrophoresis.

The charge isoform distribution of the 150 mg/mL lots was similar tothat of the 100 mg/mL lots, with only slight differences in the relativeamounts of the pE/VHS (150 mg/mL lots=31% pE/VHS; 100 mg/mL lots=34 to42% pE/VHS) and Q/VHS (150 mg/mL lots=11 to 12% Q/VHS; 100 mg/mL lots=4to 9% Q/VHS) N-terminal isoforms. The characteristics of DAC HYP are asfollows:

N-Terminal Isoforms by CEX:

pE/pE: 31-46%pE/Q: 7-12%pE/VHS: 31-42%

Q/VHS: 3-12% VHS/VHS: 1-17% C-Terminal Isoforms by CEX: 0K: 53-80% 1K:14-28% 2K: 5-19%

The N-linked glycan distribution of DAC HYP is as follows:

G0-GlcNAc: 7.2-14.6% G0: 80.9-88.2% Peak 3: 1.3-1.7% G1: 1.4-3.8

The oxidation levels measured for DAC HYP were low.

DAC HYP is biologically active, as confirmed in ELISA and surfaceplasmon resonance CD25 binding experiments, as well as functional toinhibit IL-2 induced T-cell proliferation. DAC HYP is also characterizedby a low level of aggregation upon storage.

7.7. DAC HYP Stability

High concentration DAC HYP formulations are stable upon storage. Thefollowing tables provide stability data for 150 mg/mL DAC HYP drugsubstance lots.

Table 31 below provides stability data following storage in 50 mL bagsat the recommended conditions (2-8° C.). Table 32 below providesaccelerated stability data storage in 50 mL bags at 23-27° C. Table 33below provides stressed stability data.

TABLE 31 Primary Real-Time Stability of DAC HYP Drug Substance, 150mg/mL (Batch A) Time Point Color and SEC % SEC % Binding Functional(Months) Appearance pH Conc. CEX Main Peak Aggregate % Purity* PotencyPotency 0 Pass^(a) 6.0 148 Pass^(b) 99.4 0.6 97.3 86 101 1 Pass 6.0 149Pass 99.0 1.0 97.0 90 94 2 Pass 6.0 149 Pass 99.0 1.0 96.9 91 108 3 Pass6.0 149 Pass 98.9 1.1 97.3 102 104 6 Pass 6.0 149 Pass 98.8 1.2 97.5 9479 9 Pass 6.0 151 Pass 98.5 1.4 97.3 107 110 12 Pass 6.0 152 Pass 98.41.5 95.7 96 98 ^(a)Pass criteria: Colorless, clear to slightlyopalescent liquid, essentially free of visible particles. ^(b)Passcriteria: Chromatogram profile consistent with reference. *SDS-PAGE(colloidal blue stain)

TABLE 32 Primary Accelerated Stability of DAC HYP Drug Substance, 150mg/mL (Batch A) Time Point Color and SEC % SEC % Binding Functional(Months) Appearance pH Conc. CEX Main Peak Aggregate % Purity* PotencyPotency 0 Pass^(a) 6.0 148 Pass^(b) 99.4 0.6 97.3 86 101 1 Pass 6.0 150Pass 98.5 1.4 97.0 93 111 2 Pass 6.0 152 Pass 98.3 1.5 96.0 110 105 3Pass 6.0 153 Pass 98.2 1.6 96.0 100 108 6 Pass 6.0 155 Pass 97.7 1.895.2 94 109 ^(a)Pass criteria: Colorless, clear to slightly opalescentliquid, essentially free of visible particles. ^(b)Pass criteria:Chromatogram profile consistent with reference. *SDS-PAGE (colloidalblue stain)

TABLE 33 Primary Stressed Stability of DAC HYP Drug Substance, 150 mg/mL(Batch A) Time Point Color and SEC % SEC % Binding Functional (Months)Appearance pH Conc. CEX Main Peak Aggregate % Purity* Potency Potency 0Pass 6.0 148 Pass 99.4 06 97.3 86 101 1 Pass 6.0 153 Pass 97.8 18 94.885 93 2 Pass 6.0 159 Pass 97.1 22 90.0 102 108 ^(a)Pass criteria:Colorless, clear to slightly opalescent liquid, essentially free ofvisible particles. ^(b)Pass criteria: Chromatogram profile consistentwith reference. *SDS-PAGE (colloidal blue stain)

7.8. Comparison Between Different Forms of Daclizumab

Hoffman-La Roche, Inc. (“Roche”) manufactured an intravenous formulationof a daclizumab marketed as ZENAPAX™ for treatment of allograftrejection that has been discontinued. DAC Penzberg is a 100 mg/mlsubcutaneous formulation of daclizumab used in clinical trials by PDLBioPharma (see CHOICE study described in Section 7.9.1 below).

A comparison between the DAC HYP, ZENAPAX DAC and DAC Penzbergformulations is shown in Table 34. In the table, the formulation bufferis the buffer the DAC was diafiltered into to yield the ultimateformulation. Accordingly, the noted concentrations are nominalconcentrations:

TABLE 34 DAC HYP vs. ZENAPAX DAC and DAC Penzberg Material FormulationConc. (mg/mL) ZENAPAX DAC 67 mM sodium phosphate 5 79 mM sodium chloride0.02% polysorbate 80 pH 6.9 DAC Penzberg 40 mM succinate 100 DAC HYP 100mM sodium chloride 100 or 150 0.03% polysorbate 80 pH 6.0

Various characteristics DAC HYP were compared to those of ZENAPAX DACand DAC Penzberg.

A comparison between the glycosylation of DAC HYP vs. ZENAPAX DAC isshown in FIG. 21. The analysis of exemplary lots of the three forms ofdaclizumab is set forth in Table 35:

TABLE 35 Comparison of glycosylation of DAC HYP vs. ZENAPAX DAC vs. DACPenzberg Source G0 (%) G1 (%) G2 (%) G0-GlcNAc ZENAPAX DAC 44 27 6 6 DACPenzberg 39 31 8 3 DAC HYP 84 2 <1 9

DAC HYP also has significantly lower levels of mannose glycosyls (e.g.,Man5, Man6, Man7) and lower levels of sialylated glycosyls than ZENAPAXDAC (see, e.g., FIG. 21).

Antibody dependent cell-mediated cytotoxicity (ADCC) is an in vitroassay that can be used to assess the Fc dependent activity and thepotential cytotoxic effects of antibody-target binding. Using peripheralblood mononuclear cells (PBMC) from six healthy donors as effector cellsand the CD25-expressing KIT225/K6 cell line as the target cells, theADCC activity of the various daclizumab preparations was assayed in botha variable an effector to target cell ratio format or a variableantibody concentration format.

For the variable effector to target cell ratio format, ⁵¹Cr-labeledKIT225/K6 cells (12,500 cells/well) were pre-incubated with 1 μg/mL ofantibody (final concentration) for 30 minutes at 4° C. in V-bottom96-well plates in a volume of 100 μL of ADCC Assay Medium (containing435 mL RPMI-1640; 5.0 mL L-glutamine; 50 mL heat inactivated fetalbovine serum; 500 μl 1000× 2-mercaptoethanol; 5.0 mL ofpenicillin-streptomycin (100×); and 5.0 mL of HEPES (1 M stock) per 500mL); control cells were incubated in ADCC Assay Medium in the absence ofantibody for subsequent determination of antibody-independent ⁵¹Crrelease.

The PBMC (effectors) were diluted serially in ADCC Assay Medium in aseparate 96-well polypropylene plate, yielding cell concentrations per100 μL of 6.25×10⁵ cells, 3.13×10⁵ cells, 1.56×10⁵ cells, 7.81×10⁴cells, or 3.91×10⁴ cells. A volume of 100 μL per well of PBMC suspensionwas added to the plates containing ⁵¹Cr-labeled KIT225/K6 andantibodies, yielding Effector to Target (E:T) ratios of 50:1, 25:1,12.5:1, 6.25:1 and 3.13:1. In addition, a volume of 100 μL per well ofADCC Assay Medium alone (no effector) was added to ⁵¹Cr-labeledKIT225/K6+ mAbs, to determine spontaneous release of ⁵¹Cr. The assayplates were spun at 50 RCF for 2 minutes and incubated at 37° C. in a7.5% CO₂ incubator for 4 hours.

Thirty minutes before the end of the 4-hour incubation, a volume of 25μL of 8% TritonX-100 was added to the appropriate control wells todetermine maximum release of ⁵¹Cr from target cells. Upon completion ofthe 4-hour incubation, the plates were spun at 350 RCF for 5 minutes anda volume of 100 μL of supernatant from each well was transferred tomini-tubes. Each mini-tube was inserted into a scintillation vial andcounted for 1 minute in a Beckman Gamma 5500B counter, or equivalent.

For the variable antibody concentration format, ⁵¹Cr-labeled KIT225/K6cells (12,500 cells/well; targets) were pre-incubated with various dosesof antibodies (5, 1, 0.2, 0.04, 0.008, and 0.0016 μg/mL) of mAbs (finalconcentration) for 30 minutes at 4° C. in V-bottom 96-well plates in avolume of 100 μL of ADCC Assay Medium. The control cells were incubatedwith ADCC Assay Medium alone (no mAb) for subsequent determination ofantibody-independent ⁵¹Cr release.

The PBMC (effectors) were diluted serially in ADCC Assay Medium, in aseparate 96-well polypropylene plate to a concentration of 3.13×10⁵cells/100 μL. A volume of 100 μL per well of PBMC suspension was addedto the plates containing ⁵¹Cr-labeled KIT225/K6+ mAbs, yielding anEffector to Target (E:T) ratio of 25:1. In addition, a volume of 100 μLper well of ADCC Assay Medium alone (no effector) was added to⁵¹Cr-labeled KIT225/K6+ mAbs, to determine spontaneous release of ⁵¹Cr.The assay plates were spun at 50 RCF for 2 minutes and incubated at 37°C. in a 7.5% CO₂ incubator for 4 hours.

Thirty minutes before the end of the 4-hour incubation, a volume of 25μL of 8% TritonX-100 was added to the appropriate control wells todetermine maximum release of ⁵¹Cr from target cells. Upon completion ofthe 4-hour incubation, the plates were spun at 350 RCF for 5 minutes anda volume of 100 μL of supernatant from each well was transferred tomini-tubes. Each mini-tube was inserted into a scintillation vial andcounted for 1 minute in a Beckman Gamma 5500B counter, or equivalent.

The ADCC results are shown in FIG. 22A (variable effector to target cellratio format) and FIG. 22B (variable antibody concentration format).These data show that the maximal ADCC activity achieved with DAC HYPtested at graded concentrations was approximately 30-40% lower than theactivity elicited by the same concentration of ZENAPAX DAC and DACPenzberg.

A comparison of the charge isoform profiles (corresponding to the Nterminal variants) of DAC HYP vs. ZENAPAX DAC vs. DAC Penzberg is shownin FIG. 23.

7.9. DAC HYP Clinical Trials 7.9.1. CHOICE Study

The CHOICE trial was a phase 2, randomized, double-blind,placebo-controlled trial of daclizumab added to interferon beta therapyin 230 patients with relapsing MS. The trial tested two dosing regimensof 100 mg/ml DAC Penzberg (see Section 7.8 above for a description ofthe product) administered as a subcutaneous injection: 1 mg/kgdaclizumab administered every four weeks and 2 mg/kg daclizumabadministered every two weeks. Results of the study showed that theaddition of daclizumab, administered at 2 mg/kg every two weeks tointerferon beta therapy, significantly reduced new or enlargedgadolinium-enhancing lesions at week 24, when compared to interferonbeta therapy alone.

The results of the CHOICE study are described in Wynn et al., 2010,Lancet Neurol. 9(4):381-90. Daclizumab treatment was generallywell-tolerated. Common adverse events were similar in all treatmentarms. Grade 3 adverse events were observed in 24 percent ofDAC/IFNβ-treated patients and 14 percent of placebo/IFNβ-treatedpatients. The most frequent grade 3 adverse events were infections,which occurred in 7 percent of DAC/IFNβ-treated patients and 3 percentof placebo/IFNβ-treated patients. There were no opportunistic infectionsor deaths, and all infections resolved with standard therapies.

The CHOICE trial demonstrated that, in MS patients on a background ofIFNβ-al therapy, daclizumab was well-tolerated and caused a dosedependent reduction in new/enlarged gadolinium-enhancing (Gd+) lesionsby 72% compared to IFNβ-al alone. Clinical efficacy was associated witha marked expansion of immunoregulatory CD56^(bright) natural killer (NK)cells.

7.9.2. SELECT Study

A randomized, double-blind, placebo-controlled dose ranging study(SELECT) was conducted to determine the safety and efficacy of twodifferent dosage levels of DAC HYP.

Overview.

The study was conducted at 76 centers in the Czech Republic, Germany,Hungary, India, Poland, Russia, Ukraine, and the United Kingdom. Thecare of each patient involved a treating neurologist, a treating nurse(or study coordinator), an examining neurologist, an MRI technician, anda pharmacist (or authorized designee). A centralized Interactive VoiceResponse System was used for randomization across all sites. Aprotocol-defined interim futility analysis was performed after 150patients completed the Week 24 visit.

Patients.

Eligibility criteria included patients 18-55 years of age withclinically definite relapsing remitting multiple sclerosis (according to2005 McDonald criteria #1-4; see, Polman et al, 2005 Ann Neurol58:840-846), a baseline Expanded Disability Status Scale (EDSS) of0-0.50 (Kurtzke, 1983, Neurology 33(11):1444-52) and at least one MSrelapse in the 12 months before randomization or one new Gd+ lesion onbrain MRI performed within the 6 weeks prior to randomization, wererandomized to receive either DAC HYP (150 mg or 300 mg) or placebo as asubcutaneous injection once every 4 weeks for 52 weeks. Patients withchild-bearing potential needed to practice effective contraception.

Patients were excluded if they had primary-progressive,secondary-progressive, or progressive-relapsing MS, a history ofmalignancy, severe allergic or anaphylactic reactions or known drughypersensitivity, or other significant medical conditions that, in theopinion of the investigator, would preclude administration of DAC HYP.Additional exclusion criteria included previous treatment with DAC HYPor ZENAPAX™, total lymphoid irradiation, cladribine, mitoxantrone,T-cell or T-cell receptor vaccination or any therapeutic mAb, exceptnatalizumab or rituximab. At the time of randomization, patients couldnot have received treatment with cyclophosphamide or rituximab withinthe previous year; natalizumab, cyclosporine, azathioprine,methotrexate, intravenous immunoglobulin, plasmapheresis or cytapheresiswithin the previous 6 months; or live virus vaccine, treatment withglatiramer acetate, IFNβ, interferon-alpha, 3 months beforerandomization; or corticosteroids, 4-aminopyridine or related productswithin the previous 30 days.

Characteristics of the groups were as follows:

DAC HYP Placebo 150 mg 300 mg (n = 204) (n = 208) (n = 209) DemographicsAge, years, mean (SD) 36.6 (9.0)  35.3 (8.9) 35.2 (8.7)  Gender, female,n (%) 128 (63)  140 (67) 134 (64)  Race, White, n (%) 197 (97)  202 (97)200 (96)  MS disease characteristics No prior MS therapy, n (%)* 155(76)  155 (75) 162 (78)  Years since MS diagnosis, 4.1 (2.0)  4.5 (3.0)3.7 (3.0) mean (median) Number relapses past year, 1.3 (0.6)  1.4 (0.7)1.3 (0.7) mean (SD) Baseline EDSS, mean (SD) 2.7 (1.2)  2.8 (1.2) 2.7(1.2) MRI brain lesions ≧1 Gd+ lesions, n (%)** 90 (44) 106 (51) 74 (35)No. Gd+ lesions, mean (SD)  2 (4.5)  2.1 (3.5) 1.4 (3.3) No. T2hyperintense lesions, 40 (32)  45 (35) 36 (31) mean (SD) DAC,daclizumab; HYP, high yield process; SD, standard deviation; MS,multiple sclerosis; EDSS, Expanded Disability Status Scale; Gd+,gadolinium-enhancing. *Patients who had not received prior MS treatmentwith the exception of steroids. **Placebo n = 203, DAC HYP 150 mg n =206, DAC HYP 300 mg n = 206 (all p values were >0.05 for intergroupcomparison).

Endpoints.

The primary objective of this study was to determine whether DAC HYPmonotherapy reduced MS relapses as defined by the annualized relapserate (ARR) at Week 52. Relapses were defined as new or recurrentneurologic symptoms (not associated with fever or infection),lasting >24 hours, and accompanied by new neurological findings uponassessment by the examining neurologist. An Independent NeurologyEvaluation Committee (INEC), consisting of three blinded MSneurologists, evaluated all suspected relapses to adjudicate whether theprotocol definition of MS relapse was satisfied. Only INEC approvedrelapses were included in the primary analysis.

The secondary objectives were to determine whether DAC HYP was effectivein reducing the number of cumulative new Gd+ lesions on brain MRI scansperformed at Weeks 8, 12, 16, 20 and 24 in a subset of patients;reducing the number of new or newly-enlarging T2 hyperintense lesions atWeek 52; reducing the proportion of relapsing patients between baselineand Week 52; and improving quality of life (QoL), as measured by thechange from baseline in the 29-item Multiple Sclerosis Impact Scale(MSIS-29) (Hobart et al., 2001, Brain 124(Pt 5):962-73) physical impactscore at Week 52. Confirmed disability progression was assessed bychange in EDSS score between baseline and Week 52 (1.0-point increase inEDSS for baseline EDSS ≧1.0 or 1.5 point increase for baseline EDSS=0that was sustained for 12 weeks). EDSS evaluations were conducted every12 weeks, and at Weeks 20, 52, 60 and 72.

Additional QoL endpoints were the subject's global assessment of wellbeing, as assessed by the EQ-Visual Analogue Scale (EQ-VAS) (EuroQol-anew facility for the measurement of health-related quality of life,2011, Accessed 17.11.11, at http://www.euroqol.org/); and change in theEQ-5D health survey (EuroQol-a new facility for the measurement ofhealth-related quality of life, 2011, Accessed 17.11.11, athttp://www.euroqol.org/); 12-item short form health survey SF-12 (Wareet al., 1996, Medical Care 34(3):220-33) and the MSIS-29 psychologicalscale at Week 52 (Hobart et al., 2001, Brain 124(Pt 5):962-73).

Additional MRI endpoints were the number of Gd+ lesions at Week 52, thevolume of total and new or newly enlarging T2 hyperintense lesions atWeeks 24 and 52, the volume of total and new T1 hypointense lesions“black holes” (defined as lesions that were iso/hypointense to graymatter and that did not enhance after gadolinium administration) atWeeks 24 and 52, and the percentage change in whole brain volumeassessed by the SIENA method (Smith et al., 2001, J Comput Assist Tomogr25(3):466-75).

Lymphocyte subsets were measured at multiple time points using avalidated FACS assay. CD56^(bright) NK cells were defined asCD3⁻/CD16⁺/CD56^(bright) lymphocytes. Immunogenicity to DAC HYP wasassessed using a standard ELISA to screen for anti-drug antibodies and acellular assay was then used to test for neutralizing antibodies on allpositive samples.

Statistical Analyses.

A sample size of approximately 600 patients was selected to have 90%power to detect a 50% reduction in the ARR between a DAC HYP treatmentgroup and the placebo group, estimated from simulations assuming anegative binomial distribution with a 10% drop out rate, a 5% type 1error rate and a two sided test. The ARR in the placebo group wasassumed to be 0.476, based on recently completed trials in RRMSsubjects. All reported p-values are two-tailed.

The primary analysis evaluated differences in the ARR between each DACHYP group versus placebo. Relapses that occurred after rescue treatmentwith alternative MS medication were censored. The difference wasevaluated using a negative binomial regression model adjusting for thenumber of relapses in the year before study entry, baseline EDSS (EDSS≦2.5 versus EDSS >2.5) and baseline age (≦35 versus >35 years).Secondary analyses tested for treatment differences using negativebinomial regression (number of new Gd+ lesions between weeks 8 and 24;number of new or newly enlarging T2 hyperintense lesions), a Coxproportional hazards model (time to first relapse, time to diseaseprogression), and an analysis of variance model (change in EDSS, volumeof new or newly enlarging T2 lesions, volume of new T1 hypointenselesions, QoL) and a proportional odds model (number of new Gd+ lesionsat Week 52). The proportion of patients who were relapse-free wasestimated from the Kaplan-Meier survival curve distribution.

For the cumulative number of new Gd+ lesions between Weeks 8 and 24, ifa patient missed 1 or 2 consecutive scans, or all scans, the lastnon-baseline observation was carried forward, or the mean number oflesions within each treatment group was used, respectively. For otherMRI endpoints, missing data was imputed using the mean within thetreatment group. For MSIS-29, if the patient was missing <10 items, themean of the non missing items was used to impute the score. For patientsmissing ≧10 items and for other QoL measures, a random slope andintercept model was used to estimate missing data.

Statistical testing for efficacy endpoints utilized separate comparisonsof the DAC HYP 300 mg group versus placebo and the DAC HYP 150 mg groupversus placebo. A sequential closed testing procedure was used tocontrol the overall Type I error rate due to multiple comparisons.

Efficacy analyses were evaluated in the intent-to-treat (ITT) populationwhich included all patients who underwent randomization. However, 21patients from a single study center were prospectively excluded from theITT population prior to study completion due to evidence of incorrectdosing at the center, which was identified prior to study completion(allpatients at the center were receiving active treatment). In asensitivity analysis, all primary and secondary efficacy analyses wererepeated using all randomized patients. All safety analyses were basedon the safety population, which was defined as all patients who receivedat least one dose of study medication and who had at least one postrandomization assessment.

A preplanned futility analysis was performed after 150 subjectscompleted the Week 24 visit, to provide an opportunity to stop if thehypothesized effects of DAC HYP were not evident. Since efficacy maychange over the duration of the study there was no plan to stop thestudy early for evidence of superiority at the time of the futilityanalysis. Futility was assessed by estimating separately the conditionalpower for both the cumulative number of new Gd+ lesions between weeks 8and 24 and the ARR endpoint for each dose group. The Safety MonitoringCommittee reviewed the data at the time of the analysis and based on theoverall consistency of the data and the assessment of risk benefitrecommended to continue the study.

Summary Results.

Eligible participants were randomized from Feb. 15, 2008 to May 14,2010. Baseline characteristics were similar across the three treatmentgroups, although there was a trend for patients in the DAC HYP 150 mggroup to have more T2 and Gd+T1 lesions than those in the DAC HYP 300 mggroup. Across all randomized patients, a total of 577 (93%) completedthe treatment period with similar proportions of DAC HYP andplacebo-treated patients completing the study.

Detailed Results. Clinical Efficacy.

The ARR at 52 weeks (primary endpoint) was lower for patients randomizedto DAC HYP 150 mg (0.21) or 300 mg (0.23), compared with placebo (0.46;Table 36), representing a 54% reduction versus placebo with DAC HYP 150mg (95% CI, 31% to 69%, p<0.0001), and a 50% reduction versus placebofor DAC HYP 300 mg (95% CI, 26% to 66%, p=0.0002; Table 36). Over 52weeks, the proportion of relapsing patients was reduced in the DAC HYP150 mg (19%) and 300 mg (20%) groups relapsed versus 36% in the placebogroup (p<0.001 for both comparisons) (Table 36). Compared with placebo,the risk of 3-month sustained disability progression at Week 52 wasreduced by 57% (Hazard ratio=0.43; 95% CI, 0.21 to 0.88; p=0.021) in theDAC HYP 150 mg and by 43% (Hazard ratio=0.57; 95% CI, 0.30 to 1.09;p=0.091) in the DAC HYP 300 mg group.

A relative 4.0 improvement in the MSIS-29 physical score at Week 52 wasobserved for DAC HYP 150 mg versus placebo with a less marked change inthe DAC HYP 300 mg patients, (p<0.0008 and p=0.1284 vs. placebo,respectively; Table 36). Similar improvements on other measures ofhealth-related quality of life including measures of both physical andpsychological function and overall health were also observed (Table 36).

TABLE 36 Clinical and MRI End Points by Treatment Group DAC HYP P ValuePlacebo 150 mg 300 mg DAC HYP 150 DAC HYP 300 (n = 196) (n = 201) (n =203) mg vs. Placebo mg vs. Placebo Clinical Number of Relapses  0 127(65) 163 (81) 163 (80)  1 52 (27) 33 (16) 33 (16)  2 15 (8) 5 (2) 5 (2) 3 2 (1) 0 1 (<1) ≧3 0 0 0 <0.0001 0.0002 ARR over 52 weeks (95% CI)0.46 (0.37-0.57) 0.21 (0.16-0.29) 0.23 (0.17-0.31) Rate ratio (95% CI)*0.46 (0.32-0.67) 0.50 (0.35-0.72) <0.0001 0.0002 % patients who relapsedat 52 weeks 36 19 20 Hazard ratio (95% CI)† 0.45 (0.30-0.67) 0.50(0.35-0.72) <0.0001 0.0003 Disability progression at 3-months, % 13.35.9 7.8 Rate ratio (95% CI)†† 0.43 (0.21-0.88) 0.57 (0.30-1.09) 0.0210.0905 Mean change EDSS from baseline to wk 52 0.09 −0.08 0.05 0.01020.4874 MRI New Gd+ lesions between Weeks 8-24 # patients with data¶ 104101 102 Mean no. (95% CI)|| 4.8 (3.6-6.4) 1.5 (1.1-2.0) 1.0 (0.7-1.5)<0.0001 <0.0001 % reduction versus placebo (95% CI) 69 (52.4-80.4) 78(66-86.4) New Gd+ lesions at Week 52 No. of patients with data 195 199200 Mean no. 1.4 0.3 0.2 <0.0001 <0.0001 Odds Ratio (95% CI) 0.15(0.09-0.25) 0.12 (0.07-0.20) <0.0001 <0.0001 New/newly enlarging T2hyperintense lesions at Week 52 # patients 195 199 200 Mean no. (95%CI)** 8.1 (6.7-9.9) 2.4 (2.0-3.0) 1.7 (1.4-2.2) <0.0001 <0.0001 %reduction versus placebo, (95% CI) 70 (59.4-77.9) 79 (71.3-84.2)Percentage change from baseline in volume T2 hyperintense lesions atWeek 52 # patients 193 198 197 Mean (SD)** 27.3 (107.8) −11.1 (12.1)−12.5 (12.5) <0.0001 <0.0001 Percentage change from baseline in volumeof new T1 hyperintense lesions at Week 52 # patients 195 199 200 Mean(SD)** 218.7 (400.2) 116.7 (276.6) 54.8 (153.1) <0.0001 <0.0001Percentage mean change in whole brain volume to Week 24 # patients 194198 200 Mean (SD)∞ −0.32 (0.729) −0.41 (0.769) −0.31 (0.678) 0.06350.6261 Percentage mean change in whole brain volume Week 24 to Week 52 #patients 194 198 200 Mean (SD)∞ −0.74 (0.90) −0.79 (0.83) −0.70 (0.91)0.3263 0.4117 Quality of life MSIS-29, change from baseline to Week 52,mean (SD)§ Physical Impact Score‡ 3.0 (13.5) −1.0 (11.8) 1.4 (13.5)0.0008 0.1284 Psychological Impact Score 0.6 (14.4) −1.8 (15.8) −0.5(15.3) 0.0683 0.4338 EQ-Visual Analogue Scale, change from −1.8 (13.2)2.9 (13.3) 1.0 (12.8) <0.0001 0.0149 baseline to Week 52, mean (SD)§EQ-5D Summary Health Index, change from −0.04 (0.20) 0.01 (0.18) −0.02(0.20) 0.0091 0.3538 baseline to Week 52, mean (SD)§ SF-12, change frombaseline to Week 52, mean (SD)§ Physical component −0.4 (7.0) 1.2 (7.3)0.5 (7.3) 0.0116 0.1018 Mental component −1.4 (9.2) 0.7 (9.6) −0.1 (8.6)0.0118 0.2342 ARR, annualized relapse rate; MSIS, Multiple SclerosisImpact Scale; Gd+, gadolinium-enhancing. *P values estimated from anegative binomial regression model adjusted for number of relapses in1-year period prior to study entry, baseline EDSS (≦2.5 vs. >2.5), andage (≦35 vs. >35). †P value estimated from Cox-proportional hazardsmodel adjusting for number of relapses in 1-year prior to entry,baseline EDSS, and age. ‡Lower scores indicate improvement. §P valuecalculated using analysis of covariance for difference between treatmentgroups, controlling for baseline score. ¶MRI substudy, N = 307 (placebo,n = 104; DAC HYP 150 mg, n = 101; DAC HYP 300 mg, n = 102). ||P valueestimated from a negative binomial model adjusted for the baselinenumber of Gd+ lesions. **P value estimated from a negative binomialmodel adjusted for the baseline number of T2 lesions. ††P valueestimated from Cox-proportional hazards model adjusting for baselineEDSS and age. ΔP value estimated from an analysis of covariance modeladjusted for baseline EDSS. ‡‡P value estimated from a proportional oddsmodel adjusted for the baseline number of Gd+ lesions. ∞P-value based onanalysis of covariance on ranked data adjusted for baseline normalizedbrain volume.

MRI.

DAC HYP reduced new MS lesion activity, as defined by MRI, in both theentire study population and a subset with monthly MRIs performed betweenweeks 8 to 24 (Table 36). In contrast to the clinical endpoints, thepoint estimates of efficacy were marginally stronger in the 300 mg dosegroup compared to the 150 mg dose group even after adjustment for thepotential baseline imbalances. Longitudinal analysis demonstrated thatGd+ lesion activity was higher in the 150 mg dose group compared to the300 mg dose group in the first few months of treatment but was similarby week 52. (Table 36). Sensitivity analyses that included the 21patients from the one excluded study site yielded similar results forall efficacy analyses.

Safety.

Adverse events (AEs) occurred in a similar proportion of patients in theDAC HYP 150 mg (73%), DAC HYP 300 mg (76%) and placebo (79%) groups(Table 37). Serious AEs, occurred in 26% of the placebo, 15% in the DACHYP 150 mg and 17% in the DAC HYP 300 mg groups. Excluding MS relapses,SAEs occurred in 6%, 7% and 9% of patients in each group (Table 37). AEsthat occurred in >5% of DAC HYP patients are shown in Table 37. Theincidence of serious infections was 2% in DAC HYP-treated patientsversus 0% in placebo. Among the 7 patients who had a serious infectionwhile dosing was ongoing, 1 discontinued treatment due to the seriousinfection and 6 restarted treatment after the infection resolved. Theincidence of cutaneous events was 18% in the DAC HYP 150 mg, 22% in theDAC HYP 300 mg, and 13% in the placebo groups (Table 37). Seriouscutaneous events occurred in 1% of DAC HYP-treated patients. One DACHYP-treated patient who was recovering from a serious rash died due to acomplication of a psoas abscess. At autopsy, a psoas abscess, which hadbeen previously undiagnosed, was found to involve a mesenteric arteryand had resulted in local thrombosis and acute ischemic colitis. Fivemalignancies occurred during the trial: two cases of cervical carcinoma(1 each in the placebo and DAC HYP 150 mg group); one case of thyroidneoplasm in the DAC HYP 150 mg group was a non-serious thyroid nodule;and two cases of melanoma in the DAC HYP 300 mg group. The cases ofmelanoma were treated with local excision without reported recurrence.

TABLE 37 Adverse Events Summary DAC HYP DAC HYP Placebo 150 mg 300 mg (n= 204) (n = 208) (n = 209) Adverse event summary Any adverse event, n(%) 161 (79) 151 (73) 159 (76) Any serious adverse event, 53 (26) 32(15) 36 (17) n (%) Any serious adverse event 12 (6) 15 (7) 19 (9)excluding MS relapse, n (%) Death, n 0 1* 0 Common Adverse Events thatOccurred in ≧5% of DAC HYP Patients During Treatment MS Relapse, n (%)76 (37) 43 (21) 41 (20) Nasopharyngitis, n (%) 31 (15) 30 (14) 29 (14)Upper respiratory tract 14 (7) 18 (9) 21 (10) infection, n (%) Headache,n (%) 20 (10) 18 (9) 20 (10) Pharyngitis, n (%) 8 (4) 13 (6) 13 (6)Adverse events of interest Infections, n (%) 89 (44) 104 (50) 112 (53)Serious Infections, n (%) 0 6 (3) 3 (1) Cutaneous events, n (%) 27 (13)38 (18) 45 (22) Injection-site reaction, 3 (1) 4 (2) 4 (2) erthyma,Severe cutaneous events, 0 2 (<1) 2 (1) n (%) Malignancy, n (%) 1 (<1) 2(<1) 2 (<1) Incidence of ALT Abnormalities 1-3x ULN, n (%) 64 (31) 54(26) 62 (30) 3-5x ULN, n (%) 6 (3) 7 (3) 6 (3)  >5x ULN, n (%) 1 (<1) 9(4) 8 (4) ULN, upper limit of normal; ALT, alanine aminotransferase

Laboratory Findings.

Patients treated with DAC HYP had an increase in total NK cell count(cells/mm³) compared with placebo at Week 52 (median: 42.0 (150 mg DACHYP); 46.5 (300 mg DAC HYP); vs −4.5 placebo; p=<0.001). The increase intotal NK cell numbers was related to a selective increase inCD56^(bright) NK cells from a median of 7.77 at baseline to 44.84 at endof treatment. In contrast, there were only marginal changes inCD56^(dim) NK cells (median changes from 122.68 to 123.70). Expansion ofCD56^(bright) NK cells was apparent at the first post-baseline timepoint (Week 4) in both DAC HYP arms versus placebo (p<0.0001).CD56^(bright) NK cells expanded from a median of 0.6% of lymphocytes atbaseline to 2.8% at Week 52. In contrast, patients treated with DAC HYPhad a modest decrease in B-cell and total lymphocyte counts (Table 38).Both CD4⁺ and CD8⁺ T-cell counts decreased by approximately 7-10% atWeek 52 in DAC HYP-treated patients and the CD4⁺/CD8⁺ ratio remainedconstant during treatment.

TABLE 38 Changes in Lymphocyte Cell Counts Over 52 Weeks DAC HYP Placebo150 m g 300 mg (n = 179) (n = 184) (n = 186) Total lymphocytes, meancells/mm³ (SD) Baseline 1420.9 (450.3) 1444.1 (441.9)  1395.7 (471.4) Week 24 1455.7 (468.7) 1380.9 (426.4)  1314.7 (366.3)  % Change 4.81(29.48) −1.30 (27.60)  −1.2 (29.01) Week 24 Week 52 1397.2 (414.4)1332.5 (398.4)  1286.2 (446.8)  % Change 1.89 (26.60) −3.63 (27.95)−3.69 (27.74) Week 52 B cells, mean cells/mm³ (SD) Baseline 174.4 (91.9)175.5 (89.8)  167.2 (89.6)  Week 24 187.4 (110.3) 169.4 (107.2) 150.9(94.9)  % Change 17.77 (69.77) −1.19 (42.49) −3.35 (53.29) Week 24 Week52 177.2 (81.2) 157.2 (92.9)  141.0 (76.9)  % Change 12.57 (47.25) −4.25(43.67) −11.26 (38.15)  Week 52 NK cells, mean cells/mm³ (SD) Baseline163.4 (76.7) 166.2 (110.9) 175.0 (95.7)  Week 24 169.0 (80.3) 199.3(106.6) 205.1 (87.9)  % Change 9.50 (44.88) 32.71 (56.55) 33.03 (65.13)Week 24 Week 52 158.3 (82.7) 213.0 (113.9) 223.5 (117.9) % Change 2.77(43.07) 48.14 (82.09) 50.5 (84.1) Week 52 CD4+ cells, mean cells/mm³(SD) Baseline 686.9 (248.3) 698.8 (241.3) 664.4 (261.0) Week 24 692.8(235.3) 646.2 (216.2) 606.5 (222.8) % Change 3.87 (26.72) −4.42 (27.20)−4.66 (28.24) Week 24 Week 52 682.9 (219.6) 612.8 (202.6) 581.7 (259.8)% Change 2.77 (26.45) −6.95 (30.33) −8.94 (29.97) Week 52 CD8+ cells,mean cells/mm³ (SD) Baseline 355.3 (156.2) 361.4 (146.4) 353.4 (165.8)Week 24 363.9 (176.8) 328.0 (139.6) 309.5 (133.4) % Change 4.11 (40.70)−4.73 (30.67) −6.49 (30.23) Week 24 Week 52 344.9 (160.9) 315.7 (139.6)295.7 (151.0) % Change 3.05 (44.60) −9.12 (30.96) −9.83 (33.94) Week 52CD4/CD8 ratio Baseline 2.2 (0.9) 2.1 (0.9) 2.1 (09)  Week 24 2.2 (0.9)2.2 (0.9) 2.2 (1.0) % Change 4.23 (25.38)  2.87 (18.55)  4.07 (19.85)Week 24 Week 52 2.3 (1.0) 2.2 (0.9) 2.2 (0.9) % Change 6.36 (30.00) 4.57 (18.77)  4.47 (20.63) Week 52 SD, standard deviation; NK, naturalkiller.

Liver function test (LFT) abnormalities were above 5×ULN, occurred in 4%of DAC- and <1% of placebo-treated patients. These abnormalitiestypically occurred late in the treatment period (median onset+day 308)and resolved with a median time of 62 days. Of the 17 DAC HYP-treatedpatients with elevations of >5×ULN, 6 continued or resumed treatmentwith DAC HYP for at least 6 months after resolution, all withoutrecurrence during this period. In 2 patients, LFT elevations wereassociated with infections (one case of hepatitis B and one case ofcytomegalovirus infection).

Immunogenicity.

At week 24, neutralizing antibodies to DAC HYP were detected in 6 (2%)DAC HYP-treated patients (5 patients in the 150 mg dose group and 1subject in the 300 mg dose group). In some patients these antibodieswere transient, and at week 52 neutralizing antibodies to DAC HYP werepresent in only 1 subject from each DAC HYP dose group.

Conclusion.

Antagonism of CD25 with monthly, subcutaneous DAC HYP monotherapydemonstrated robust and clinically meaningful effects over 1 year on MSdisease activity, e.g., as measured by reduction in relapse rate, newMRI defined lesion activity and disability progression in apredominantly treatment naïve population of MS patients.

8. SPECIFIC EMBODIMENTS, INCORPORATION BY REFERENCE

Various aspects of the present disclosure are described in theembodiments set forth in the following numbered paragraphs.

1. A modified NS0 cell that has been adapted to grow in serum- andcholesterol-free media and that is engineered to express a recombinantprotein, said cell being capable of achieving a volumetric productivityexceeding 100 mg/L/day recombinant protein in a culture of 100 L in a10-day fed-batch process when grown in serum- and cholesterol-freemedia.2. The modified NS0 cell of embodiment 1 which is capable of achieving avolumetric productivity exceeding 100 mg/L/day recombinant protein in aculture of 1,000 L in a 10-day fed-batch process when grown in mediafree of cholesterol and animal-derived components.3. The modified NS0 cell of embodiment 1 which is capable of achieving avolumetric productivity exceeding 100 mg/L/day recombinant protein in aculture of 16,000 L in a 10-day fed-batch process when grown in mediafree of cholesterol and animal-derived components.4. The modified NS0 cell of any one of embodiments 1-3 to which a feedmedium is added according to the following schedule, where the volumeadded represents the percentage of the initial cell culture volume:

Day Volume added 0 0 1 0 2 4 3 7.8 4 7.8 5 7.8 6 11 7 13 8 15 9 15 10 05. The modified NS0 cell of embodiment 1 that is capable of achieving avolumetric productivity exceeding 200 mg/L/day recombinant protein in aculture of at least 100 L in a 13-day fed-batch process.6. The modified NS0 cell of embodiment 5 which is capable of achieving avolumetric productivity exceeding 200 mg/L/day recombinant protein in aculture of 1,000 L in a 13-day fed-batch process when grown incholesterol-free media.7. The modified NS0 cell of embodiment 5 which is capable of achieving avolumetric productivity exceeding 100 mg/L/day recombinant protein in aculture of 16,000 L in a 10-day fed-batch process when grown in serum-and cholesterol-free media.8. The modified NS0 cell of embodiment 1 which is stably transfectedwith a nucleic acid useful for expressing an anti-CD25 monoclonalantibody.9. The modified NS0 cell of embodiment 8 in which the anti-CD25monoclonal antibody comprises a VL chain corresponding in sequence topositions 21-233 of SEQ ID NO:2 and a VH chain corresponding in sequenceto positions 20 to 465 of SEQ ID NO:4.10. The modified NS0 cell of embodiment 1 which was transformed withvector pAbX.gpt.11. The modified NS0 cell of embodiment 1 which was transformed withvector pHAT.IgG1.rg.dE.12. The modified NS0 cell of embodiment 1 which is designated as clone7A11-5H7-14-43.13. A method of producing a recombinant protein, comprising culturingthe modified NS0 cell of any one of embodiments 1-12.14. The method of embodiment 13, wherein the modified NS0 cell iscultured under conditions that result in the production of at least 100mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture ina 10-day fed-batch process, or at least 200 mg/L/day recombinant proteinin a 100 L, 1,000 L or 16,000 L culture in a 13-day fed-batch process.15. The method of embodiment 13 or embodiment 14, wherein the modifiedNS0 cell is cultured in the absence of serum and cholesterol.16. The method of embodiment 15 wherein the modified NS0 cell iscultured in the absence of tropolone and hydrocortisone.17. The method of embodiment 13 or embodiment 14, wherein the modifiedNS0 cell is cultured in a basal and/or feed medium containing 10-35 g/Lglucose.18. The method of embodiment 17, wherein the modified NS0 cell iscultured in a basal medium containing 15 g/L glucose and/or a feedmedium containing 28 g/L glucose.19. The method of embodiment 18, wherein the basal medium is composed ofthe components of PFBM2±10%.20. The method of embodiment 18, wherein the feed medium is composed ofthe components of PFFM3±10%.21. The method of embodiment 19 or embodiment 20, wherein the cell iscultured in basal medium for 1-3 days, and then in feed medium for 10-13days.22. The method of embodiment 18, wherein the feed medium is addedaccording to the schedule outlined in Table 7±10%.23. A vector useful for recombinantly expressing a protein of interest,comprising a weak promoter driving expression of a selectable markeroperable in mammalian cells and a strong promoter driving expression ofa protein of interest.24. The vector of embodiment 23, wherein the protein of interest is atherapeutic antibody.25. The vector of embodiment 24, wherein the therapeutic antibody is ananti-CD25 antibody.26. The vector of embodiment 25, wherein the anti-CD25 antibodycomprises the CDRs of daclizumab.27. The vector of embodiment 26, wherein the anti-CD25 antibody isdaclizumab.28. A method for obtaining a mammalian host cell that has a highvolumetric productivity of a protein of interest, comprisingtransfecting the cell with the vector of any one of embodiments 23-27,and selecting a cell that is capable of producing at least 100 mg/L/dayprotein of interest in a 100 L, 1,000 L or 16,000 L culture in a 10-dayfed-batch process or at least 200 mg/L/day recombinant protein in a 100L, 1,000 L or 16,000 L culture in a 13-day fed-batch process.29. A composition comprising daclizumab, where the daclizumab ischaracterized by the presence of a pE/Q heavy chain N-linked isoformand/or a Q/VHS heavy chain N-terminal isoform.30. The composition of embodiment 29 in which the pE/Q heavy chainN-terminal isoform constitutes approximately 6-15% of the daclizumab.31. The composition of embodiment 29 in which the pE/Q heavy chainN-terminal isoform constitutes approximately 7-12% of the daclizumab.32. The composition of any one of embodiments 29-31 in which the Q/VHSheavy chain N-terminal isoform constitutes approximately 1-15% of thedaclizumab.33. The composition of embodiment 32 in which the Q/VHS heavy chainN-terminal isoform constitutes approximately 3-12% of the daclizumab.34. The composition of embodiment 29 in which the heavy chain ofdaclizumab exists in the following N-terminal isoforms:

Isoform Prevalence pE/pE 25%-50% pE/Q  6%-15% pE/VHS 25%-48% Q/VHS 1%-15% VHS/VHS 0.5%-25% 35. The composition of embodiment 29 in which the heavy chain ofdaclizumab exists in the following N-terminal isoforms:

Isoform Prevalence pE/pE  31-46% pE/Q 7%-12% pE/VHS 31%-42%  Q/VHS3%-12% VHS/VHS 2%-17%36. The composition of embodiment 29, where the daclizumab ischaracterized by a cation exchange chromatography isoform profilesubstantially similar to that of FIG. 18.37. The composition of embodiment 29, where the daclizumab is DAC HYP.38. A composition comprising daclizumab, where the daclizumab ischaracterized by an N-linked glycosylation HPLC profile containing twomain peaks, one corresponding to oligosaccharide G0 GlcNAc and onecorresponding to oligosaccharide G0, where the combined AUC of these twopeaks constitutes about 88-99.5% of the total AUC of all peaks.39. The composition of embodiment 38, in which the AUC of the G0 GlcNAcpeak constitutes about 5-18% of the total AUC of all peaks and the AUCof the G0 peak constitutes about 75-92% of the total AUC of all peaks.40. The composition of embodiment 39, in which the AUC of the G0-GlcNAcpeak constitutes about 6-16% of the total AUC of all peaks and the AUCof the G0 peak constitutes about 78-90% of the total AUC of all peaks.41. The composition of any one of embodiments 38-40 in which theN-linked glycosylation profile has less than about 3% of Man5.42. The composition of embodiment 41 in which the N-linked glycosylationprofile has less than about 0.5% G2, Man6 and/or Man7.43. The composition of embodiment 38, in which the N-linkedglycosylation profile contains a third peak corresponding to sialylatedoligosaccharides, and the AUC of the sialylated oligosaccharide peakconstitutes 1% or less of the total AUC of all peaks.44. The composition of embodiment 38, in which the N-linkedglycosylation profile contains a third peak corresponding tooligosaccharide G1, and the AUC of the G1 peak constitutes about 1-5% ofthe total AUC of all peaks.45. The composition of embodiment 44, in which the AUC of the G1 peakconstitutes about 1-2% of the total AUC of all peaks.46. The composition of embodiment 38, in which the daclizumab has anN-linked glycosylation HPLC profile substantially similar to that ofFIG. 19 or to that of the lower panel of FIG. 21.47. The composition of embodiment 38, in which the daclizumab is DACHYP.48. A composition comprising daclizumab which exhibits less than 35%ADCC average cytotoxicity as measured in an in vitro cellular assayusing effector cells from at least 3 healthy donors and Kit 225 K6 cellsas target cells, at a daclizumab concentration of 1 μg/mL and aneffector to target cell ratio of about 25:1.49. The composition of embodiment 48 wherein the daclizumab exhibits 10%to 30% ADCC average cytotoxicity in said assay.50. The composition of embodiment 48 or embodiment 49 wherein said assayuses effector cells from at least 6 healthy donors.51. The composition of embodiment 48 or embodiment 49 wherein said assayuses effector cells from at least 10 healthy donors.52. The composition of any one of embodiments 48-51, in which thedaclizumab is DAC HYP.53. A composition useful for making a daclizumab drug formulation,comprising about 150-190 mg/mL daclizumab and quantities of excipientssuch that dilution of the composition with a dilution buffer yields adiluted composition that contains about 85-165 mg/mL daclizumab and hasan osmolality in the range of about 267-327 mOsm/kg and a pH in therange of about pH 5.8-6.2 at 25° C., and in which at least about 95% ofthe daclizumab is in monomer form, as measured by size exclusionchromatography.54. The composition of embodiment 53 which contains quantities ofexcipients such that when diluted with a dilution buffer the dilutedcomposition contains about 85-115 mg/mL daclizumab.55. The composition of embodiment 53 which contains quantities ofexcipients such that when diluted with a dilution buffer the dilutedcomposition contains about 150±15 mg/mL daclizumab.56. A composition comprising about 4 to 15 mg/mL daclizumab, where 0.1%or less of the daclizumab is in aggregate form.57. The composition of embodiment 56 which is obtained by purifying adaclizumab composition comprising about 4 to 15 mg/mL daclizimab, whereup to 2.5% of the daclizumab is in aggregate form, via columnchromatography on a weak cation exchange resin.58. The composition of embodiment 57, where the weak cation exchangeresin is CM-650M.59. The composition of embodiment 58, where the CM-650M resin isequilibrated with an equilibration buffer containing about 20 mM sodiumcitrate, pH 4.4-4.6, and the daclizumab is eluted with an elution buffercontaining about 20 mM sodium citrate and about 75 mM sodium sulfate, pH4.4-4.6.60. The composition of embodiment 59, where the chromatography iscarried out in a cylindrical column using a resin bed having a height ofabout 10-30 cm or about 17-19 cm, and the daclizumab is eluted at atemperature in the range of about 4-22° C. or about 18-22° C., and aflow rate in the range of about 50-200 cm/hr or about 90-110 cm/hr.61. A composition suitable for administration to humans, comprisingabout 85-165 mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate80, where the composition has an osmolality in the range of about267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., andat least about 95% of the daclizumab is in monomer form, as measured bysize exclusion chromatography.62. The composition of embodiment 61, in which at least about 99% of thedaclizumab is in monomer form, as measured by size exclusionchromatography.63. The composition of embodiment 61 which comprises about 85-115 mg/mLdaclizumab.64. The composition of embodiment 63, which consists essentially ofabout 100 mg/mL daclizumab, about 40 mM sodium succinate, about 100 mMsodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH ofabout 6.0 at 25° C.65. The composition of embodiment 61 which comprises about 135-165 mg/mLdaclizumab.66. The composition of embodiment 65, which consists essentially ofabout 150 mg/mL daclizumab, about 40 mM sodium succinate, about 100 mMsodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH ofabout 6.0 at 25° C.67. The composition of embodiment 65 which is obtained by a processcomprising the steps of concentrating a daclizumab compositioncomprising about 4 to 15 mg/mL daclizumab via ultrafiltration in asuitable buffer to achieve a daclizumab concentration in the range ofabout 85-180 mg/mL and optionally diluting the concentrated compositionwith a dilution buffer.68. A pharmaceutical composition suitable for subcutaneousadministration comprising about 85-165 mg/mL daclizumab, where thepercentage of daclizumab in aggregate form does not exceed about 3%following storage for a period of about 12 months at a temperature inthe range of about 2-8° C.69. The pharmaceutical composition of embodiment 68 which comprisesabout 85-115 mg/mL daclizumab.70. The pharmaceutical composition of embodiment 68 which comprisesabout 135-165 mg/mL daclizumab.71. The pharmaceutical composition of embodiment 69 or embodiment 70 inwhich the percentage of daclizumab in aggregate form does not exceedabout 2% following storage for a period of about 12 months at atemperature in the range of about 2-8° C.72. The pharmaceutical composition of embodiment 69 or embodiment 70 inwhich the percentage of daclizumab in aggregate form does not exceedabout 3% following storage for a period of about 18 months at atemperature in the range of about 2-8° C.73. A process for harvesting a recombinant protein from a cell culture,comprising the steps of:

-   -   (i) adjusting the pH of a cell culture that expresses and        secretes a recombinant protein to a pH in the range of about pH        4.5-5.5;    -   (ii) incubating the pH-adjusted cell culture for about 30-90        minutes at a temperature in the range of about 4 to 15° C.; and    -   (iii) centrifuging the incubated pH-adjusted cell culture to        remove cell debris.        74. A process for producing a purified daclizumab composition,        comprising the steps of:    -   (i) adsorbing daclizumab from a crude daclizumab preparation        onto affinity chromatography resin;    -   (ii) washing the affinity chromatography resin with a wash        buffer to remove contaminants;    -   (iii) eluting the adsorbed daclizumab with an elution buffer;    -   (iv) inactivating viruses in the eluate by adjusting the pH to a        pH in the range of about pH 3-4 and incubating the pH-adjusted        eluate at specified temperature for a period of time sufficient        to inactivate viruses;    -   (v) neutralizing the virus-inactivated eluate to a pH in the        range of about pH 7.7-7.9 (measured at 25° C.);    -   (vi) flowing the neutralized eluate across a strong anion        exchange chromatography resin;    -   (vii) adsorbing the daclizumab of the eluate of step (vi) onto a        weak cation exchange chromatography resin; and    -   (viii) eluting the adsorbed daclizumab from the weak cation        exchange chromatography resin.        75. The process of embodiment 74 in which the crude daclizumab        preparation is harvested from a cell culture.        76. The process of embodiment 74 in which the crude daclizumab        preparation is obtained by culturing host cell 7A11-5H7-14-43        under conditions in which daclizumab is secreted into the        culture medium and harvesting the secreted daclizumab.        77. The process of embodiment 76 in which the daclizumab is        harvested using the method of embodiment 73.        78. The process of embodiment 74, which further comprises the        steps of:    -   (ix) filtering the eluted daclizumab composition of step (viii)        to remove viruses; and    -   (x) concentrating the filtered solution via ultrafiltration to        yield a purified, daclizumab composition comprising about 85-180        mg/mL daclizumab.        79. The process of embodiment 78, which further comprises the        step of diluting the purified daclizumab composition with a        dilution buffer so as to obtain a composition comprising about        85-165 mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate        80, where the composition has an osmolality in the range of        about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2        at 25° C., and at least about 95% of the daclizumab is in        monomer form, as measured by size exclusion chromatography.        80. The process of embodiment 79, wherein the composition        obtained has less than 50 ppm of host cell proteins from a        recombinant source of daclizumab, less than 10 ppm of protein A,        and no more than 3% of the daclizumab in the composition is in        aggregate form.        81. Basal medium PFBM2.        82. Feed medium PFFM3.        83. A daclizumab composition, which is obtained by a process        comprising the step of culturing a host cell according to any        one of embodiments 1-12 under conditions in which daclizumab is        secreted into the culture medium.        84. The daclizumab composition of embodiment 83, in which the        process further comprises the step of isolating the secreted        daclizumab from the cell culture medium.        85. A buffer useful for sanitizing a protein A affinity        chromatography resin, comprising about 100-500 mM sodium        citrate, about 10-30 mM NaOH and about 0.5-3% (v/v) benzyl        alcohol.        86. A method of sanitizing a protein A affinity chromatography        column, comprising washing the column with the sanitization        buffer of embodiment 85 at a flow rate and for a period of time        sufficient to sanitize the column.        87. The method of embodiment 86, in which the column is washed        with approximately 1.8 column volumes of sanitization buffer at        a flow rate of about 150 cm/hr, the washed column incubated        without flow for a period of about 30-45 min., and then        equilibrated with an equilibration buffer.        88. The method of embodiment 87, in which the equilibration        buffer comprises about 20 mM sodium citrate and 150 mM NaCl, and        has a pH of about pH 7 (at 25° C.).        89. A method of treating a patient suffering from multiple        sclerosis, comprising administering to the patient an amount of        a DAC HYP composition sufficient to provide therapeutic benefit.        90. The method of embodiment 89 in which the DAC HYP composition        is administered intravenously.        91. The method of embodiment 90 in which the DAC HYP composition        is administered in amount corresponding to about 0.8-0.9 mg/kg        DAC HYP.        92. The method of embodiment 91 in which the DAC HYP composition        is administered in an amount corresponding to about 1 mg/kg DAC        HYP.        93. The method of any one of embodiments 89-92 in which the DAC        HYP is administered once per week for a period of at least 6        weeks, at least 12 weeks, at least 24 weeks.        94. The method of any one of embodiments 89-93 in which the DAC        HYP is administered as monotherapy.        95. The method of embodiment 94 in which the patient has either        failed to respond to prior treatment with interferon-beta or has        discontinued prior treatment with interferon-beta.        96. The method of any one of embodiments 89-93 in which the DAC        HYP is administered adjunctively to interferon-beta.        97. The method of embodiment 89 in which the DAC HYP composition        is administered subcutaneously.        98. The method of embodiment 97 in which the DAC HYP composition        is administered in an amount corresponding to about 1 mg/kg DAC        HYP.        99. The method of embodiment 98 in which the DAC HYP composition        is administered once every two weeks.        100. The method of embodiment 99 in which the DAC HYP        composition is administered for a total of about 24 weeks.        101. The method of embodiment 97 in which the DAC HYP        composition is administered in an amount corresponding to about        2 mg/kg DAC HYP.        102. The method of embodiment 101 in which the DAC HYP        composition is administered once every 4 weeks.        103. The method of embodiment 102 in which the DAC HYP        composition is administered for a total of about 24 weeks.        104. The method of embodiment 103, in which the DAC HYP        composition is administered in an amount corresponding to 75 mg        to 300 mg DAC HYP.        105. The method of embodiment 104 in which the DAC HYP        composition is administered in an amount corresponding to 150        mg.        106. The method of embodiment 104 in which the DAC HYP        composition is administered in an amount corresponding to 300        mg.        107. The method of any one of embodiments 103-106 wherein the        DAC HYP composition is administered once every 4 weeks.        108. The method of embodiment 107 wherein the DAC HYP        composition is administered for a total of at least 48 weeks.        109. The method of any one of embodiments 103-108 in which the        DAC HYP is administered as monotherapy.        110. The method of embodiment 109 in which the patient has        either failed to respond to prior treatment with interferon-beta        or has discontinued prior treatment with interferon-beta.        111. The method of any one of embodiments 103-108 in which the        DAC HYP is administered adjunctively to interferon-beta.        112. A recombinant daclizumab, from a cell culture, obtained or        obtainable by a process comprising the steps of:    -   (i) adjusting the pH of a cell culture that expresses and        secretes a recombinant protein to a pH in the range of about pH        4.5-5.5;    -   (ii) incubating the pH-adjusted cell culture for about 30-90        minutes at a temperature in the range of about 4 to 15° C.; and    -   (iii) centrifuging the incubated pH-adjusted cell culture to        remove cell debris.        113. A purified daclizumab composition, obtained or obtainable        by a process comprising the steps of:    -   (i) adsorbing daclizumab from a crude daclizumab preparation        onto affinity chromatography resin;    -   (ii) washing the affinity chromatography resin with a wash        buffer to remove contaminants;    -   (iii) eluting the adsorbed daclizumab with an elution buffer;    -   (iv) inactivating viruses in the eluate by adjusting the pH to a        pH in the range of about pH 3-4 and incubating the pH-adjusted        eluate at specified temperature for a period of time sufficient        to inactivate viruses;    -   (v) neutralizing the virus-inactivated eluate to a pH in the        range of about pH 7.7-7.9 (measured at 25° C.);    -   (vi) flowing the neutralized eluate across a strong anion        exchange chromatography resin;    -   (vii) adsorbing the daclizumab of the eluate of step (vi) onto a        weak cation exchange chromatography resin; and    -   (viii) eluting the adsorbed daclizumab from the weak cation        exchange chromatography resin.        114. A purified daclizumab composition, obtained or obtainable        by the process of any one of embodiments 74-80.

Deposit of strain: A strain of NS0 cells adapted to grow in serum-freeand cholesterol-free medium that has been stably transfected with vectorpHAT.IgG1.rg.dE and which can be used to produce DAC HYP, clone7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911, wasdeposited with the American Type Tissue Collection (“ATCC”) at 10801University Blvd., Manassas, Va. 20510-209, U.S.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1.-15. (canceled)
 16. A pharmaceutical composition suitable forsubcutaneous administration comprising about 135-165 mg/mL daclizumab,wherein the daclizumab has one or more of the following: (a) anN-terminal isoform with one pyroglutamate residue and one glutamineresidue that comprises 3%-17% of the total daclizumab; (b) an N-linkedoligosaccharide profile comprising two main peaks and a minor peak,wherein one of the two main peaks is G0-GlcNAc glycoform present inabout 5% to about 20% of the AUC, the other main peak is G0 glycoformpresent in about 70% to about 99.2% of the AUC, and the minor peak is G1glycoform present in 1% to 9% of the AUC when determined according tothe method described in Section 7.6.14. 17.-33. (canceled)
 34. Thepharmaceutical composition of claim 16, comprising about 150 mg/mLdaclizumab.
 35. The pharmaceutical composition of claim 16, furthercomprising sodium succinate.
 36. The pharmaceutical composition of claim35, wherein the sodium succinate is present in about 5.9 mg/mL.
 37. Thepharmaceutical composition of claim 16, further comprising succinicacid.
 38. The pharmaceutical composition of claim 37, wherein thesuccinic acid is present in about 0.4 mg/mL.
 39. The pharmaceuticalcomposition of claim 16, further comprising sodium chloride.
 40. Thepharmaceutical composition of claim 39, wherein the sodium chloride ispresent in about 5.8 mg/mL.
 41. The pharmaceutical composition of claim16, further comprising polysorbate
 80. 42. The pharmaceuticalcomposition of claim 41, wherein the polysorbate 80 is present in about0.3 mg/mL.
 43. The pharmaceutical composition of claim 1, wherein the pHis about pH 6.0.
 44. A pharmaceutical composition suitable forsubcutaneous administration comprising about 150 mg/mL daclizumab,wherein the daclizumab has one or more of the following: (a) anN-terminal isoform with one pyroglutamate residue and one glutamineresidue that comprises 3%-17% of the total daclizumab; (b) an N-linkedoligosaccharide profile comprising two main peaks and a minor peak,wherein one of the two main peaks is G0-GlcNAc glycoform present inabout 5% to about 20% of the AUC, the other main peak is G0 glycoformpresent in about 70% to about 99.2% of the AUC, and the minor peak is G1glycoform present in 1% to 9% of the AUC when determined according tothe method described in Section 7.6.14; and wherein the pharmaceuticalcomposition further comprises: about 5.9 mg/mL sodium succinate, about0.4 mg/mL succinic acid, about 5.8 mg/mL sodium chloride, and about 0.3mg/mL polysorbate 80, and wherein the pharmaceutical composition has apH of about pH 6.0.